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Brown adipose tissue (BAT) generates heat through non-shivering thermogenesis, and increasing BAT amounts or activity could facilitate obesity treatment and provide metabolic benefits. In mice, BAT has been reported in perirenal, thoracic and cranial sites. Here, we describe new pelvic and lower abdominal BAT depots located around the urethra, internal reproductive and urinary tract organs and major lower pelvic blood vessels, as well as between adjacent muscles where the upper hind leg meets the abdominal cavity. Immunohistochemical, western blot and PCR analyses revealed that these tissues expressed BAT markers such as uncoupling protein 1 (UCP1) and CIDEA, but not white adipose markers, and β3-adrenergic stimulation increased UCP1 amounts, a classic characteristic of BAT tissue. The newly identified BAT stores contained extensive sympathetic innervation with high mitochondrial density and multilocular lipid droplets similar to interscapular BAT. BAT repositories were present and functional neonatally, and showed developmental changes between the neonatal and adult periods. In summary, several new depots showing classical BAT characteristics are reported and characterized in the lower abdominal/pelvic region of mice. These BAT stores are likely significant metabolic regulators in the mouse and some data suggests that similar BAT depots may also exist in humans.

Signaling by androgens through androgen receptor (AR) is essential to complete spermatogenesis in the testis. Similarly, loss of the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), results in male infertility, due in part to indirect deleterious effects on the seminiferous epithelium and spermatogenesis. Effects of steroid hormones are induced primarily through genomic changes induced by hormone-mediated activation of their intracellular receptors and subsequent effects on nuclear gene transcription. However, androgens and estrogens also signal through rapid nonclassical pathways involving actions initiated at the cell membrane. Here we review the data that nonclassical androgen and estrogen signaling pathways support processes essential for male fertility in the testis and reproductive tract. The recent development of transgenic mice lacking nonclassical AR or ESR1 signaling but retaining genomic nuclear signaling has provided a powerful tool to elucidate the function of nonclassical signaling in the overall response to androgens and estrogens. Results from these mice have emphasized that nonclassical signaling is essential for full responses to these hormones, and absence of either nonclassical or classical AR or ESR1 pathways produces abnormalities in spermatogenesis and the male reproductive tract. Although additional work is required to fully understand how classical and nonclassical receptor signaling synergize to produce full steroid hormone responses, here we summarize the known physiological functions of the classical and nonclassical androgen and estrogen signaling pathways in the testis and reproductive tract.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that suppresses gene expression. Previously, we developed a conditional null model where EZH2 is knocked out in uterus. Deletion of uterine EZH2 increased proliferation of luminal and glandular epithelial cells. Herein, we used RNA-Seq in wild-type (WT) and EZH2 conditional knockout ( Ezh2cKO) uteri to obtain mechanistic insights into the gene expression changes that underpin the pathogenesis observed in these mice. Ovariectomized adult Ezh2cKO mice were treated with vehicle (V) or 17β-estradiol (E2; 1 ng/g). Uteri were collected at postnatal day (PND) 75 for RNA-Seq or immunostaining for epithelial proliferation. Weighted gene coexpression network analysis was used to link uterine gene expression patterns and epithelial proliferation. In V-treated mice, 88 transcripts were differentially expressed (DEG) in Ezh2cKO mice, and Bmp5, Crabp2, Lgr5, and Sprr2f were upregulated. E2 treatment resulted in 40 DEG with Krt5, Krt15, Olig3, Crabp1, and Serpinb7 upregulated in Ezh2cKO compared with control mice. Transcript analysis relative to proliferation rates revealed two module eigengenes correlated with epithelial proliferation in WT V vs. Ezh2cKO V and WT E2 vs. Ezh2cKO E2 mice, with a positive relationship in the former and inverse in the latter. Notably, the ESR1, Wnt, and Hippo signaling pathways were among those functionally enriched in Ezh2cKO females. Current results reveal unique gene expression patterns in Ezh2cKO uterus and provide insight into how loss of this critical epigenetic regulator assumingly contributes to uterine abnormalities.

Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multiorgan autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient ((−/−)) mice, Aire(−/−) or wild-type (WT) males were paired with WT females. Aire(−/−) males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire(−/−) mice were defective in their ability to fertilize WT oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire(−/−) male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland, and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as in the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.

Mouse penile development is androgen-dependent. During development of male and female external genitalia, an internal ectodermal epithelial structure forms called the preputial lamina. At puberty the male preputial lamina canalizes to create the preputial space, effectively splitting into two layers: (a) the epithelial lining of the prepuce and (b) the surface epithelium of the penis. The female preputial lamina does not canalize, and instead remodels into the inverted U-shaped clitoral lamina of the adult female mouse. Androgen-dependent penile development was studied in transgenic mice with pathway-selective AR mutant transgenes through which AR signaling was activated either via the classical (AR-C) or the nonclassical pathway (AR-NC). Penile development and canalization of the preputial lamina was observed in AR-C and wild-type male mice naturally having both AR-C and AR-NC pathways. Conversely, clitoral development occurred in AR null (lacking both AR-C and AR-NC pathways) and AR-NC mice. The process of canalization of the preputial lamina seen in wild-type, AR-C and AR-C/AR-NC male mice involved cornification of the preputial lamina which involved up-regulation of keratin 10 and loricrin. Such up-regulation of these epidermal proteins was absent in the developing and adult clitoral lamina seen in wild-type female mice and AR-NC and AR null male (XY) mice. Thus, signaling through AR-C is sufficient to initiate and promote penile development and canalization of the preputial lamina, a process involving epithelial cornification.

Both membrane and nuclear fractions of estrogen receptor 1 (ESR1) mediate 17β-estradiol (E2) actions. Mice expressing nuclear (n)ESR1 but lacking membrane (m)ESR1 (nuclear-only estrogen receptor 1 [NOER] mice) show reduced E2 responsivity and reproductive abnormalities culminating in adult male and female infertility. Using this model, we investigated whether reproductive pathologies caused by the synthetic estrogen diethylstilbestrol (DES) are mitigated by mESR1 ablation. Homozygous and heterozygous wild-type (WT and HET, respectively) and NOER male and female mice were subcutaneously injected with DES (1 mg/kg body weight [BW]) or vehicle daily from postnatal day (PND) 1–5. Uterine histology was assessed in select DES-treated females at PND 5, whereas others were ovariectomized at PND 60 and treated with E2 (10 μg/kg BW) or vehicle 2 weeks later. Neonatal DES exposure resulted in ovary-independent epithelial proliferation in the vagina and uterus of WT but not NOER females. Neonatal DES treatment also induced ovary-independent adult expression of classical E2-induced transcripts (e.g., lactoferrin [Ltf] and enhancer of zeste homolog 2 [Ezh2]) in WT but not NOER mice. At PND 90, DES-treated WT and HET males showed smaller testes and a high incidence of bacterial pyogranulomatous inflammation encompassing the testes, epididymis and occasionally the ductus deferens with spread to lumbar lymph nodes; such changes were largely absent in NOER males. Results indicate that male and female NOER mice are protected from deleterious effects of neonatal DES, and thus mESR1 signaling is required for adult manifestation of DES-induced reproductive pathologies in both sexes.

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.

The common view on penile development is that it is androgen-dependent, based first and foremost on the fact that the genital tubercle forms a penis in males and a clitoris in females. However, critical examination of the complex processes involved in human penile development reveals that many individual steps in development of the genital tubercle are common to both males and females, and thus can be interpreted as androgen-independent. For certain developmental events this conclusion is bolstered by observations in androgen-insensitive patients and androgen receptor mutant mice. Events in genital tubercle development that are common to human males and females include: formation of (a) the genital tubercle, (b) the urethral plate, (c) the urethral groove, (d) the glans, (e) the prepuce and (f) the corporal body. For humans 6 of 13 individual developmental steps in penile development were interpreted as androgen-independent. For mice 5 of 11 individual developmental steps were found to be androgen-independent, which were verified through analysis of androgen-insensitive mutants. Observations from development of external genitalia of other species (moles and spotted hyena) provide further examples of androgen-independent events in penile development. These observations support the counter-intuitive idea that penile development involves both androgen-independent and androgen-dependent processes.

The role of tissue interactions was explored to determine whether epithelial differentiation within the developing human reproductive tract is induced and specified by mesenchyme in tissue recombinants composed of mouse vaginal mesenchyme + human uterine tubal epithelium (mVgM+hTubE). The tissue recombinants were grown in DES-treated ovariectomized athymic mice. After 2 to 4 weeks of in vivo growth, several vaginal specific features were expressed in the human tubal epithelium. The mesenchyme-induced effects included morphological change as well as expression of several immunohistochemical markers. Although the mesenchyme-induced shift in vaginal differentiation in the human tubal epithelium was not complete, the partial induction of vaginal markers in human tubal epithelium verifies the importance of mesenchymal-epithelial interactions in development of the human female reproductive tract. In a separate experiment, DES-induction of uterine epithelial progesterone receptor (PGR) and estrogen receptor 1 (ESR1) was explored in tissue recombinants composed of wild-type or Esr1KO mouse uterine mesenchyme + human fetal uterine epithelium (wt UtM+hUtE and Esr1KO UtM+hUtE). The rationale of this experiment was to determine whether DES-induction of PGR and ESR1 is mediated directly via epithelial ESR1 or indirectly (paracrine mechanism) via mesenchymal ESR1. DES-induction of uterine epithelial ESR1 and PGR in Esr1KO UtM+hUtE tissue recombinants (devoid of mesenchymal ESR1) formally eliminates the paracrine mechanism and demonstrates that DES induction of human uterine epithelial ESR1 and PGR is directly mediated via epithelial ESR1.

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 17β-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.

Molecular mechanisms by which androgens signal through the androgen receptor (AR) to maintain male fertility are poorly understood. Transgenic mice were produced expressing mutant ARs that can only (1) alter gene transcription through the classical response pathway (AR-C) or (2) activate kinase signaling cascades via the nonclassical pathway (AR-NC). AR-C is sufficient to produce sperm and fertility. Haploid germ cell production, the blood-testis barrier, and spermatid migration are supported by AR-NC. Gene expression essential for chromosome synapsis during meiosis requires AR-C. We identify targets of androgen signaling required for male fertility and provide a mechanistic explanation for meiotic germ cell arrest in the absence of androgen signaling. Prostate differentiation occurs with AR-C alone, but full development requires synergistic nonclassical signaling. Both AR signaling pathways are necessary for normal male reproductive tract development and function, validating our mouse models for studies of AR functions in other target tissues.

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. 17β-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.

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation. The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12 to P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0 to P10 period. In “estrogen mutant mice” (NERKI, βERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17β-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.Summary SentenceLoss of the histone methyltransferase EZH2 in the mouse uterus results in changes in uterine development and ovary-independent epithelial proliferation and ultimately causes infertility.

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17β-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.

Estrogen binding to estrogen receptors (ESR) triggers signaling cascades within cells. Historically, a major emphasis has been characterizing estrogen-induced genomic actions resulting from binding to nuclear estrogen receptor 1 (nESR1). However, recent evidence indicates the first receptors estrogens encounter as they enter a cell, membrane ESR1 (mESR1), also play crucial roles. Membrane and nuclear ESR are derived from the same transcripts but the former are directed to the membrane via palmitoylation. Binding and activation of mESR1 leads to rapid fluctuations in cAMP and Ca(+2) and stimulation of protein kinase pathways. Endocrine disrupting chemicals (EDC) that mimic 17β-estradiol can signal through mESR1 and elicit non-genomic effects. Most current EDC studies have focused on genomic actions via nESR1. However, increasing number of studies have begun to examine potential EDC effects mediated through mESR1, and some EDC might have higher potency for signaling through mESR1 than nESR1. The notion that such chemicals might also affect mESR1 signaling via palmitoylation and depalmitoylation pathways has also begun to gain currency. Recent development of transgenic mice that lack either mESR1 or nESR1, while retaining functional ESR1 in the other compartment, will allow more precise in vivo approaches to determine EDC effects through nESR1 and/or mESR1. It is increasingly becoming apparent in this quickly evolving field that EDC directly affect mESR and estrogen signaling, but such chemicals can also affect proportion of ESR reaching the membrane.Future E D C studies should be designed to consider the full range of effects through mESR alone and in combination with nESR.

There have been significant breakthroughs over the past decade in the development and use of pluripotent stem cells as a potential source of cells for applications in regenerative medicine. It is likely that this methodology will begin to play an important role in human clinical medicine in the years to come. This review describes the plasticity of one type of pluripotent cell, spermatogonial stem cells (SSCs), and their potential therapeutic applications in regenerative medicine and male infertility. Normally, SSCs give rise to sperm when in the testis. However, both human and murine SSCs can give rise to cells with embryonic stem (ES) cell-like characteristics that can be directed to differentiate into tissues of all three embryonic germ layers when placed in an appropriate inductive microenvironment, which is in contrast to other postnatal stem cells. Previous studies have reported that SSCs expressed an intermediate pluripotent phenotype before differentiating into a specific cell type and that extended culture was necessary for this to occur. However, recent studies from our group using a tissue recombination model demonstrated that SSCs differentiated rapidly into another tissue, in this case, prostatic epithelium, without expression of pluripotent ES cell markers before differentiation. These results suggest that SSCs are capable of directly differentiating into other cell types without going through an intermediate ES cell-like stage. Because SSCs do not require reprogramming to achieve a pluripotent state, they are an attractive source of pluripotent cells for use in regenerative medicine.

The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13–16) in stages I–VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized with H+-ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility.

Testes contain two distinct Leydig cell populations during development: fetal and adult Leydig cells (FLCs and ALCs, respectively). ALCs are not derived from FLCs, and it is unknown whether these two populations share common progenitors. We discovered that hedgehog (Hh) signaling is responsible for transforming steroidogenic factor 1-positive (SF1+) progenitors into FLCs. However, not all SF1+ progenitors become FLCs, and some remain undifferentiated through fetal development. We therefore hypothesized that if FLCs and ALCs share SF1+ progenitors, increased Hh pathway activation in SF1+ progenitor cells could change the dynamics and distribution of SF1+ progenitors, FLCs, and ALCs. Using a genetic model involving constitutive activation of Hh pathway in SF1+ cells, we observed reduced numbers of SF1+ progenitor cells and increased FLCs. Conversely, increased Hh activation led to decreased ALC populations prepubertally, while adult ALC numbers were comparable to control testes. Hence, reduction in SF1+ progenitors temporarily affects ALC numbers, suggesting that SF1+ progenitors in fetal testes are a potential source of both FLCs and ALCs. Besides transient ALC defects, adult animals with Hh activation in SF1+ progenitors had reduced testicular weight, oligospermia, and decreased sperm mobility. These defects highlight the importance of properly regulated Hh signaling in Leydig cell development and testicular functions.—Barsoum, I. B., Kaur, J. Ge, R. S., Cooke, P. S., Yao, H. H.-C. Dynamic changes in fetal Leydig cell populations influence adult Leydig cell populations in mice.

Mesoangiobasts are blood vessel-derived stem cells that differentiate into smooth, skeletal, and cardiac muscle cells. We have reported that postnatal aorta-derived mesoangioblasts (ADM) regenerate skeletal muscle and prevent onset of dilated cardiomyopathy in animal models of Duchenne muscular dystrophy. ADM also differentiate into myelinating glial cells, suggesting they are multipotent and capable of generating mesodermal or ectodermal derivatives. Mesenchyme of some fetal organs is a potent instructive inducer. Here we examined whether ADM can differentiate into prostatic, uterine, and skin epithelium by recombining ADM with fetal or neonatal mesenchyme from these organs and grafting them under the renal capsule of syngeneic hosts. In tissue recombinants of uterine mesenchyme (UtM) and ADM, ADM formed histologically normal simple columnar uterine epithelium that expressed estrogen receptor 1 and in response to estrogen showed increased mitogenesis and downregulation of progesterone receptor. In contrast, ADM did not differentiate into prostatic epithelium or epidermis when recombined with urogenital sinus mesenchyme or fetal dermis, respectively. These results indicate that ADM can respond to cues from neonatal UtM and differentiate into morphologically and functionally normal uterine epithelial cells, and support previous reports that ADM can differentiate into a variety of tissues of the mesodermal lineage. However, these data indicate that ADM are restricted in their capacity to differentiate into endodermal and ectodermal derivatives such as prostatic and skin epithelial cells, respectively.

During neonatal and juvenile life, mammalian uteri undergo extensive structural and functional changes, including uterine gland differentiation and development. In sheep and mice, inhibition of neonatal uterine gland development induced by progestin treatment led to a permanent aglandular uterine phenotype and adult infertility, suggesting that this strategy might be useful for sterilizing dogs and other companion animals. The goal of this study was to define temporal patterns of adenogenesis (gland development), cell proliferation, and progesterone and estrogen receptor expression in uteri of neonatal and juvenile dogs as a first step toward determining whether neonatal progestin treatments might be a feasible contraceptive approach in this species. Uteri obtained from puppies at postnatal wk 1, 2, 4, 6, or 8 were evaluated histologically and immunostained for MKI67, a marker of cell proliferation, estrogen receptor-1, and progesterone receptor. Adenogenesis was under way at 1 wk of age, as indicated by the presence of nascent glands beginning to bud from the luminal epithelium, and rapid proliferation of both luminal epithelial and stromal cells. By Week 2, glands were clearly identifiable and proliferation of luminal, glandular, and stromal cells was pronounced. At Week 4, increased numbers of endometrial glands were evident penetrating uterine stroma, even as proliferative activity decreased in all cell compartments as compared with Week 2. Whereas gland development was most advanced at Weeks 6 to 8, luminal, glandular, and stromal proliferation was minimal, indicating that the uterus was nearly mitotically quiescent at this age. Both estrogen receptor-1 and progesterone receptor were expressed consistently in uterine stromal and epithelial cells at all ages examined. In summary, canine uterine adenogenesis was underway by 1 wk of age and prepubertal glandular proliferation was essentially complete by Week 6. These results provided information necessary to facilitate development of canine sterilization strategies based on neonatal progestin treatments designed to permanently inhibit uterine gland development and adult fertility.

The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10–13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.

Soy-based infant formulas are widely used in the United States and some other countries. These formulas contain high levels of the estrogenic isoflavone genistein, leading to concern that neonatal genistein exposure could cause acute and/or long-term adverse effects on reproductive and other organs. However, previous work to assess genistein effects in rodent models has not typically replicated the route of delivery and/or serum genistein concentrations reported for soy formula-fed human infants. Our objective was to develop a mouse model that more closely mimics the oral genistein exposure and total serum genistein concentrations observed in soy formula-fed infants. Mouse pups were dosed orally with genistein in a soy formula-corn oil emulsion from Postnatal Day (PND) 1 to PND 5, then effects on reproductive and non-reproductive organs were assessed after dosing and during subsequent development. Neonatal treatment resulted in changes both at the completion of dosing (PND 5) and in adult animals. At PND 5, neonatal genistein treatment caused increased relative uterine weight and down-regulation of progesterone receptor in uterine epithelia. Estrogenic effects of genistein were also seen in the neonatal ovary and thymus, which had an increase in the incidence of multioocyte follicles (MOFs) and a decrease in thymic weight relative to body weight, respectively. The increased incidence of MOFs persisted into adulthood for neonatally treated genistein females, and estrous cycle abnormalities were seen at 6 mo of age despite normal fertility in these mice. The immediate and long-term effects in this neonatal animal model raise concerns that high serum concentrations of genistein are estrogenic and could potentially impact the development of human infants fed soy formula.

The blood-testis barrier (BTB) is formed by tight junctions between Sertoli cells. Results of previous studies suggested that the barrier is deficient in ets variant 5 (ETV5) gene-deleted mice; therefore, microarray data were examined for changes in tight junction-associated genes. The tight junctional protein claudin 5 (CLDN5) was decreased in testes of 8-day-old Etv5(-/-) pups. The study reported herein examined the expression of CLDN5 in wild-type (WT) and Etv5(-/-) mice and evaluated its contribution to BTB function. CLDN5 protein expression was evaluated in 8-day-old WT and Etv5(-/-) and adult WT, Etv5(-/-), and W/W(v) testes by immunohistochemistry and in 8-day-old WT Sertoli cell-enriched and germ cell-enriched fractions by immunocytochemistry. Cldn5 mRNA expression was evaluated in 0- to 20-day-old and adult WT mice and in 8-day-old and adult Etv5(-/-) mice via quantitative PCR. Tracer studies were performed in adult WT, Etv5(-/-), and W/W(v) mice. The results indicate the following: 1) CLDN5 was expressed in Sertoli cells, spermatogonia, and preleptotene spermatocytes. 2) Seminiferous epithelial CLDN5 expression depended upon both the presence of germ cells and ETV5. 3) CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. 4) Cldn5 mRNA expression increased in the testes of juvenile mice at the time of BTB formation. 5) Testes of Etv5(-/-) and W/W(v) mice, which are both deficient in seminiferous epithelial CLDN5 expression, had biotin tracer leakage from the interstitial space into the seminiferous tubule lumen. In conclusion, CLDN5 is expressed in the seminiferous epithelium, appears to be regulated by multiple influences, and contributes to BTB function.

Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

Pluripotent stem cells have great clinical potential for tissue regeneration/repair in humans. The use of embryonic stem (ES) cells is ethically controversial, leading to searches for other sources of pluripotent stem cells. Testicular spermatogonial stem cells (SSCs) produce the spermatogenic lineage. Under in vitro conditions, SSCs have the ability to give rise to pluripotent ES-like cells. We hypothesized that stem/progenitor spermatogonia could directly transdifferentiate into different tissue types if they were recombined with inductive mesenchymes from fetal/neonatal organs using a tissue separation/recombination methodology and grown in vivo. Green fluorescent protein transgenic mice were used to track cell lineages. Our results indicate that stem/progenitor spermatogonia recombined with the appropriate mesenchyme can directly transdifferentiate in vivo into tissues of all germ layers, including prostatic, uterine, and skin epithelium. In addition, transdifferentiated tissue expressed molecular, histological, and functional markers of the appropriate epithelium. The ability of stem/progenitor spermatogonia to directly generate various epithelia emphasizes their clinical potential, and if adult human SSCs have similar properties, this may have applications in human regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 ( Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; 1 µg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

The objective of this study was to determine whether stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild-type (wt) and LGR7 knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep, and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17beta (designated d 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10 of treatment. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before cervices and vaginas were collected at 1000 h on d 10. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling was used to quantify apoptosis. Relaxin markedly increased proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P0.01) but had no effect on tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of cervix and vagina, whereas epithelial LGR7 does not affect these processes.

Connexin 43 (CX43, also known as GJA1) is the predominant testicular gap-junction protein. In the seminiferous epithelium, gap junctions occur between adjacent Sertoli cells and between Sertoli and germ cells. The specific role of CX43 in Sertoli cell has been difficult to study because of the neonatal lethality of the Cx43 global knockout. Recently two laboratories have independently developed a Sertoli cell conditional Cx43 knockout using Cre-loxP methodology. These have allowed the role of CX43 in Sertoli cells to be determined while circumventing lethal abnormalities in organs such as the heart seen in Cx43 global knockouts. In this review the focus is on the insights into the role of CX43 in Sertoli cells derived from these new model systems. Results indicate that CX43 is essential for cessation of proliferation and normal maturation in Sertoli cells. In addition, CX43 in Sertoli cells is required for spermatogenesis, but not early germ cell proliferation. These model systems show that CX43 is important in Sertoli cell development, and will be useful in further studies of its role in Sertoli cell and testis biology.

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFalpha and IL-1beta stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin-dependent kinase inhibitor p27 regulates activity of cyclin/cyclin-dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1-Cullin-F-box protein) complex targets proteins such as p27 for ubiquitin-proteosome degradation; the F box protein S phase kinase-associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2−/−) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild-type (WT), Skp2−/−, and p27−/−Skp2−/− mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2−/− fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2−/− mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27−/−Skp2−/− double knockout mice. The Skp2−/− decrements in adipocyte number and fat pad mass were totally reversed in p27−/−Skp2−/− mice. Adipogenesis was inhibited in MEFs from Skp2−/− vs. WT mice, and this inhibition was absent in MEFs from p27−/−Skp2−/− mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.

Previously, we reported an association between estrogen receptor-alpha (ERalpha) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERalpha knockout (ERalphaKO) mouse model to test the hypothesis that ERalpha mediates DES effects in the developing penis. ERalphaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 microg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERalphaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERalphaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERalphaKO mice when compared with controls, although testosterone concentration was much higher in the ERalphaKO mice. Hence, the resistance of ERalphaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERalpha in mediating the harmful effects of neonatal DES exposure in the developing penis.

Neonatal uterus and vagina express estrogen receptor 1 (ESR1) and respond mitogenically to exogenous estrogens. However, neonatal ovariectomy does not inhibit preweaning uterine cell proliferation, indicating that this process is estrogen independent. Extensive literature suggests that ESR1 can be activated by growth factors in a ligand-independent manner and drive uterine cell proliferation. Alternatively, neonatal uterine cell proliferation could be ESR1 independent despite its obligatory role in adult luminal epithelial proliferation. To determine ESR1's role in uterine and vaginal development, we analyzed cell proliferation, apoptosis, and uterine gland development (adenogenesis) in wild-type (WT) and Esr1 knockout (Esr1KO) mice from Postnatal Day 2 to Postnatal Day 60. Uterine and vaginal cell proliferation, apoptosis, and uterine adenogenesis were comparable in WT and Esr1KO mice before weaning. By Days 29-60, glands had regressed, and uterine cell proliferation was reduced in Esr1KO mice in contrast to continued adenogenesis and proliferation in WT. Apoptosis in Esr1KO uterine epithelium was not increased compared to WT at any age, indicating that differences in cell proliferation, rather than apoptosis, cause divergence of uterine size in these two groups at puberty. Similarly, vaginal epithelial proliferation was reduced, and the epithelium became atrophic in Esr1KO mice by 29 days of age and later in Esr1KO mice. These results indicate that preweaning uterine and vaginal development is ESR1 independent but becomes dependent on ESR1 by Day 29 on. It is not yet clear what mechanisms drive preweaning vaginal and uterine development, but ligand-independent activation of ESR1 is not involved.

Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.

Potential therapeutic use of stem cells in the treatment of human diseases depends on our ability to control the balance of their differentiation and self-renewal in vitro and in vivo. The stem cell “niche,” or specialized microenvironment, is now recognized as one of the major contributors to this regulation in many species. Our recent study, which was reported in Nature, was the first to demonstrate that expression of a vertebrate animal transcription factor is essential for the maintenance of a stem cell niche. In that letter, targeted disruption of ERM (ets-related molecule), which was localized only in the somatic support cell of the testis, the Sertoli cell, resulted in failure of self-renewal by spermatogonial stem cells, following the first wave of spermatogenesis. One of the more important conclusions drawn was the realization that regulation of the stem cell niche during the perinatal period, a phase characterized by rapid mitosis of both spermatogonial stem cells and Sertoli cells, differed from that in the adult. It appears that the ERM-regulated pathways are coincident with the termination of Sertoli cell proliferation and commencement of the cycle of spermatogenesis, which is sustained by the same cell that regulates the stem cell niche. Several likely targets for ERM regulation are discussed, as well as their potential implications for increasing our understanding of spermatogonial stem cell activity and the uniqueness of the Sertoli cell's immune privilege and possible utility for the protection of transplanted adult stem cells.

Estrogen regulates thymic development and involution and modulates immune function. Despite its critical role in thymus, as well as in autoimmune disorders, the mechanism by which estrogen affects the thymus is not well understood. We previously reported that the estrogenic soy isoflavone genistein, as well as 17beta-estradiol (E2), could induce thymic involution, but genistein effects were only partially mediated through estrogen receptors. To provide insights into mechanisms of estrogenic effects in the thymus, we investigated thymic gene expression changes induced by E2 (125 ng/day) and genistein (1500 ppm in feed) in weanling mice using high-density DNA arrays. We identified several E2-responsive genes involved in thymic development and thymocyte signaling during selection and maturation. Functional characterization indicated effects on genes involved in transcription, apoptosis, and the cell cycle. This study also identified changes in several E2-regulated transcripts essential to maintain immune self-tolerance. E2 upregulated more genes than genistein, while genistein downregulated more genes than E2. Though each treatment regulated several genes not altered by the other, there was considerable overlap in the genes regulated by E2 and genistein. Changes in transcription factors and cell cycle factors were consistent with decreases in cell proliferation induced by both genistein and E2. As indicated by the regulation of non-E2-responsive genes, genistein also induced unique effects through non-estrogenic mechanisms. The specific downregulation of the CD4 coreceptor transcript by genistein was consistent with the decline of CD4+ thymocytes in genistein-treated mice in our previous study. This is the first study identifying E2 and genistein target genes in the thymus. These findings provide new mechanistic insights toward explaining estrogen action on thymocyte development, selection, and maturation, as well as the effects of genistein on prenatal and neonatal thymic development and function.

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure inhibits ventral, dorsolateral, and anterior prostate development in C57BL/6 mice. To determine if prostatic abnormalities persist into senescence, mice born to dams given TCDD (5 mug/kg, po) or vehicle on gestation day 13 were examined at 100 and 510 days of age. Half the mice were castrated ten days prior to necropsy in order to assess androgen dependence, while the remaining mice were sham castrated. Effects of TCDD on the dorsolateral and anterior prostate of senescent sham-castrated mice were relatively subtle, whereas the ventral prostate was rudimentary or absent. Castration of vehicle-exposed mice caused far greater reductions in prostate lobe weights, epithelial cell height, and androgen-dependent gene expression (MP25 and probasin) in young mice than in senescent ones, while cell proliferation was decreased by castration in young mice and increased in senescence. Responses to castration were similar at 100 days of age in vehicle- and TCDD-exposed mice. At 510 days, however, TCDD-exposed mice were substantially more responsive to castration by most indices than vehicle-exposed mice. These results demonstrate that prostatic androgen dependence in mice declines substantially with age in several key ways, and that in utero and lactational TCDD exposure protects against this decline. Surprisingly, TCDD increased the incidence of cribriform structures in dorsolateral prostate ducts, from 2-3% in vehicle-exposed senescent mice to 16% in sham-castrated and to 7% in castrated senescent mice. Collectively, these results demonstrate that effects of in utero and lactational TCDD exposure on the prostate persist into senescence, and suggest that in utero and lactational TCDD exposure retards the aging process in the prostate. However, because cribriform structures are often considered to be associated with prostate carcinogenesis, these results also suggest that TCDD exposure early in development may increase susceptibility to prostate cancer.

Estrogen has historically been viewed as a major regulator of adipose tissue in adult females, but recent work has indicated that estrogen's role in adipose biology may be broader than initially appreciated and has also provided important insights into the mechanism of estrogen effects on adipose tissue. Estrogen has direct effects on adipocytes to inhibit lipogenesis and may also have direct effects on other cellular constituents of adipose tissue, as well as metabolic effects on other target organs that can regulate adipose tissue. Estrogen has central effects on food consumption and energy expenditure that contribute to its overall inhibitory effects on adipose deposition. Estrogen also plays an important role in regulating adipose deposition in males and recently has been shown to be an important factor in the determination of adipocyte number, indicating that it regulates key developmental events in adipogenesis. Although critical questions still remain in our understanding of the overall role of estrogen in adipose tissue, it is clear that estrogen plays a more important role in adipose tissue than originally realized and that it is a major regulator of adipose tissue in both sexes during development and adulthood.

Gender influences mediated by 17 beta-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-alpha (Esr1) is crucial for the protective effect of 17 beta-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wild-type Esr1+/+ or Esr1 knockout (Esr1-/-) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1+/+ disease-inducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in EAE.

Steroid sex hormones (17beta-estradiol, testosterone, dihydrotestosterone, and progesterone) and aryl hydrocarbons such as the dioxins regulate epithelial proliferation and secretory protein production and differentiation in their respective target organs in male and female urogenital tracts and mammary glands. Recent evidence has demonstrated that stromal-epithelial interactions are critical for mediating the effects of these molecules on epithelial cells. Our results have indicated that estradiol, testosterone, progesterone, and dioxin regulate epithelial proliferation (stimulation or inhibition) via paracrine mechanisms requiring the appropriate receptor in the stroma. The androgen receptor (AR), estrogen receptor alpha (ERalpha), progesterone receptor (PR), or aryl hydrocarbon receptor (AhR) in the epithelium are neither necessary nor sufficient for the regulation of epithelial proliferation. Moreover, during prostatic development, signaling through the stromal AR is required to induce prostatic epithelial identity, ductal morphogenesis and glandular differentiation. Epithelial functional differentiation is regulated in the prostate, uterus, and vagina via AR (prostate) and ERalpha(uterus and vagina). In these organs both epithelial and stromal steroid receptors are required for steroidal regulation of certain aspects of epithelial differentiation such as epithelial secretory protein production in the uterus and epithelial cornification in the vagina and prostate (squamous metaplasia). The mechanistic basis of these stromal-epithelial interactions is poorly understood, but growth factors appear to be mediators of these cell-cell interactions.

IGF-I is a critical regulator of uterine growth, and locally produced uterine IGF-I could mediate the effects of 17-E2 on growth and cellular proliferation. We used IGF-I knockout (KO) mice and tissue grafting to determine the roles of local and systemic IGF-I in uterine growth and E2 responsiveness. Uteri from adult KO mice and neonatal and adult wild-type (WT) mice were grown under the renal capsule of female athymic mice for 4 wk. Initial uterine weights of adult KO and neonatal WT mice were 5% or less of those of adult WT uteri. Weights of adult WT uterine grafts did not increase during grafting. Weights of adult KO and neonatal WT uteri exposed to normal systemic levels of IGF-I in athymic hosts increased 20- to 30-fold to equal or exceed those of adult WT grafts. Uterine epithelial height, reduced in KO mice, was restored to WT levels in KO uteri grafted into athymic hosts. The absence of local IGF-I production in KO uteri did not impair E2induced epithelial proliferation in KO uterine grafts. Neonatal WT uteri grafted into KO hosts showed minimal growth, providing evidence that local uterine IGF-I production is insufficient to support uterine growth in the absence of systemic IGF-I. E2 treatment of KO females produced minimal uterine growth, confirming that lack of IGF-I, rather than E2, caused the uterine hypoplasia. In summary, systemic IGF-I supports normal uterine growth and E2 response in the absence of local IGF-I. Local IGF-I is not a direct mediator of E2 action in uterus, and systemic IGF-I may be more important than previously thought for growth of the uterus and other tissues. (Endocrinology 143: 2673–2679, 2002)

Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4+CD8−and double-positive CD4+CD8+thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4+CD8−helper T cell lineage. Decreases in the relative percentages of CD4+CD8−thymocytes were accompanied by decreases in relative percentages of splenic CD4+CD8−cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.

Progesterone (P4) and the synthetic glucocorticoid dexamethasone (Dex) inhibit luminal epithelial (LE) proliferation in neonatal mouse uteri. This study determined the roles of progesterone receptor and estrogen receptor 1 (PR and ESR1, respectively) in P4- and Dex-induced inhibition of LE proliferation using PR knockout (PRKO) and Esr1 knockout (Esr1KO) mice. Wild-type (WT), heterozygous, and homozygous PRKO female pups were injected with vehicle, P4 (40 μg/g body weight), or Dex (4 or 40 μg/g body weight) on Postnatal Day 5, then 24 h later immunostained for markers of cell proliferation. In WT and heterozygous mice, P4 sharply reduced LE proliferation, and Dex produced dose-responsive decreases equaling those of P4 at the higher dose. Critically, although both doses of Dex similarly decreased proliferation compared to vehicle-treated PRKOs, treatment of PRKO pups with the high Dex dose (40 μg/g) did not inhibit LE as much as treatments of WT mice with this Dex dose or with P4. Stromal proliferation was stimulated by P4 in WT but not PRKO mice, and Dex did not alter stromal proliferation. Uteri of all genotypes strongly expressed glucocorticoid receptor (GR), demonstrating that impaired Dex effects in PRKOs did not reflect GR deficiency. Furthermore, inhibition of LE proliferation by Dex (40 μg/g body weight) in Esr1KO mice was normal, so this process does not involve ESR1. In summary, inhibitory Dex effects on LE proliferation occur partially through non-PR-mediated mechanisms, presumably GR, as indicated by Dex inhibition of LE proliferation in PRKOs. However, maximal inhibitory Dex effects on uterine LE proliferation are not seen in PRKO mice with even high Dex, indicating that maximal Dex effects in WT mice also involve PR.

Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets.

During organogenesis, the middle to caudal portion of Müllerian epithelium differentiates into uterine and vaginal epithelia in females. Functional differentiation of uterine and vaginal epithelia occurs in adulthood, and is regulated by 17beta-estradiol (E(2)) and progesterone. In this report, the roles of mesenchyme/stroma in differentiation of uterine and vaginal epithelia were studied in tissue recombination experiments. At birth, Müllerian epithelium was negative for uterine and vaginal epithelial markers. Tissue recombinant experiments showed that uterine and vaginal gene expression patterns were induced in neonatal Müllerian epithelium by the respective mesenchymes. Differentiated adult uterine and vaginal epithelia did not change their original gene expression in response to heterotypic mesenchymal induction. In the adult vagina, E(2) induced expression of involucrin, a CCAAT/enhancer-binding protein beta and cytokeratin 1 via estrogen receptor alpha (ERalpha). Tissue recombination experiments with wild-type and ERalpha knockout mice demonstrated that epithelial gene expression is regulated by E(2) via epithelial-stromal tissue interactions. Uterine/vaginal heterotypic tissue recombinations demonstrated that functional differentiation of uterine and vaginal epithelia required organ-specific stromal factors. In contrast, stromal signals regulating epithelial proliferation appeared to be nonspecific in the uterus and vagina.

Experiments were conducted to determine the role of the aryl hydrocarbon receptor (AhR) in the development of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 male mice. Male and female mice heterozygous for the AhR (Ahr+/-) were mated, and pregnant females were dosed orally on gestation day 13 with corn oil vehicle or TCDD (5 microg/kg). Pups were necropsied on postnatal day (PND) 21, 35, and 90. Comparison of vehicle-exposed wild-type (Ahr+/+) pups with vehicle-exposed AhR knockout (AhRKO; Ahr-/-) pups confirmed and extended previous reports that development of the liver, heart, spleen, thymus, and kidney is affected by absence of the AhR. Lung, submandibular gland, testis, and epididymis weights were also affected, indicating that the AhR plays a role in normal development of these organs as well. The presence or absence of the AhR had no effect on the incidence of hydronephrosis, daily sperm production, or cauda epididymal sperm numbers in vehicle-exposed mice. TCDD caused numerous effects in wild-type mice that were absent in AhRKO mice; specifically, hydronephrosis, increases in relative liver and heart weight, decreases in absolute heart and lung weight, and decreases in absolute and relative thymus, submandibular gland, epididymis, and testis weight. In several cases, TCDD produced one effect in wild-type mice (reductions in body weight and absolute thymus, submandibular gland, and epididymis weight on PND 21; and reductions in absolute and relative submandibular gland and absolute testis weight on PND 35) but caused the opposite effect in AhRKO mice. In yet other cases (reduced relative spleen weight on PND 21 and reductions in absolute and relative thymus weight on PND 35), TCDD produced similar effects in wildtype and AhRKO mice. There were also cases in which TCDD significantly affected AhRKO mice without significantly altering the same endpoint in wild-type mice; absolute liver, lung, and kidney weight were increased and relative submandibular gland weight was decreased on PND 21; relative heart weight was reduced and absolute lung weight increased on PND 35; and relative liver weight was decreased on PND 90. Although many effects of TCDD required the presence of the AhR, these results provide evidence either for multiple forms of the AhR in mice (one or more of which are still present in AhRKO mice), or for AhR-independent effects of low-level TCDD exposure.

Approximately 2 million women in the USA and Europe continue taking oral contraceptives each year during undetected pregnancy due primarily to non-compliance and also to individual variation in sensitivity to hormones in the contraceptives. Prenatal exposure to oral contraceptives containing 17alpha-ethinyl oestradiol (EE) has generally not been associated with an increased incidence of externally observable malformations at birth. The purpose of this study was to assess effects on reproductive organs in adult male mice that had been exposed during gestation day 0 through 17 (equivalent to gestation week 16 in humans) to clinically relevant (approximately 0.5 microg/kg/day) and lower doses of EE. Doses used in this study ranged from 0.002 to 2 microg/kg/day. By 5 months of age, prostate weight was significantly (P < 0.05) higher than controls in most treatment groups of EE (0.02-2 microg/kg). Prostatic androgen receptor populations were significantly elevated only in the 0.02 microg/kg group, suggesting different mechanisms for the increase in prostate weight at different doses. Daily sperm production (DSP) and DSP per gramme of testis were reduced in all treatment groups during adolescence, but not later in adulthood. These findings are consistent with prior studies showing that prenatal exposure of mice to very low doses of a number of oestrogenic chemicals can alter the adult male reproductive system without causing gross external malformations.

Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERalpha-knockout (alphaERKO) male and female mice to determine the role of ERalpha in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in alphaERKO compared with WT males; weights of various WAT depots in alphaERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in alphaERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from alphaERKO compared with WT males. Compared with WT controls, alphaERKO females also had increases in WAT. The alphaERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERalpha or aromatase. The obesity in alphaERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERalpha absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERalpha signaling system is critical in female and male WAT deposition, and may have clinical implications.

We investigated the function of estrogen receptor-α in global myocardial ischemia and reperfusion injury in male estrogen receptor-α knockout (ERKO) and wild-type mice. Mouse hearts were subjected to 45 min of global ischemia followed by 180 min of reperfusion. The hearts were excised, cannulated, and maintained in a chilled (4°C) cardioplegia solution until warm (37°C) oxygenated Krebs-Henseleit bicarbonate buffer was perfused through the coronary arteries. ERKO hearts started beating later and had a higher incidence of ventricular fibrillation and/or tachycardia than control hearts. Coronary flow rate was significantly lower in ERKO hearts during the 90- and 120-min periods of reperfusion. Ca2+ accumulation was significantly greater following 30, 90, 120, 150, and 180 min of reperfusion in ERKO hearts. Nitrite production was significantly less in ERKO hearts following 90, 120, and 150 min of reperfusion. Myocardial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was significantly lower in experimental ERKO hearts. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of ischemia-reperfused ERKO hearts but not in control tissues. Hematoxylin-basic fuchsin-picric acid-stained sections from experimental ERKO hearts had fewer viable myocytes compared with controls. Transmission electron microscopy revealed swollen and fragmented mitochondria with amorphous and granular bodies, loss of matrix, and rupture of cristae in experimental ERKO hearts. This is the first demonstration that estrogen receptor-α plays a cardioprotective role in ischemia-reperfusion injury in males.