The overexpression of the insl3 in female mice causes descent of the ovaries

Testicular descent in mice is dependent upon proper outgrowth of the gubernaculum primordia under the influence of the insulin-like 3 gene product (Insl3). Deletion of this gene prevents gubernaculum growth and causes bilateral cryptorchidism. In vitro experiments have led to the suggestion that Insl3 and androgens together induce outgrowth of the gubernacular primordia. The experiments reported here were designed specifically to determine whether in vivo the Insl3-mediated gubernaculum development is independent of androgens. To that effect transgenic male and female mice were generated that overexpressed Insl3 in the pancreas during fetal and postnatal life. Expression of the transgenic allele in the Insl3-deficient mice rescued the cryptorchidism in male mutant, indicating that the islet beta-cells efficiently processed the Insl3 gene product to the functional hormone. All transgenic females displayed bilateral inguinal hernia. The processus vaginalis developed containing intestinal loops. The Müllerian derivatives gave rise to oviduct, uterus, and upper vagina, and Wolffian duct derivatives were missing, indicating the absence of the androgen- and anti-Müllerian hormone-mediated activities in transgenic females. The ovaries descended into a position over the bladder and attached to the abdominal wall via the well developed cranial suspensory ligament and the gubernaculum. Administration of dihydrotestosterone during prenatal development suppressed formation of the cranial suspensory ligament and thereby allowed the descent of the ovaries into the processus vaginalis. These results suggest that Insl3-mediated activity induces gubernaculum development and precludes a role of androgen in this process. Furthermore, the transgenic females exhibit reduced fertility, which is due to fetal mortality during midgestation.     

GREAT/LGR8 is the only receptor for insulin-like 3 peptide

During male development testes descend from their embryonic intraabdominal position into the scrotum. Two genes, encoding the insulin-like 3 peptide (INSL3) and the GREAT/LGR8 G protein-coupled receptor, control the differentiation of gubernaculum, the caudal genitoinguinal ligament critical for testicular descent. It was established that the INSL3 peptide activates GREAT/LGR8 receptor in vitro. Mutations of Insl3 or Great cause cryptorchidism (undescended testes) in mice. Overexpression of the transgenic Insl3 causes male-like gubernaculum differentiation, ovarian descent into lower abdominal position, and reduced fertility in females. To address the question whether Great deletion complements the mutant female phenotype caused by the Insl3 overexpression, we have produced Insl3 transgenic mice deficient for Great. Such females had a wild-type phenotype, demonstrating that Great was the only cognate receptor for Insl3 in vivo. We have established that pancreatic HIT cells, transfected with the INSL3 cDNA, produce functionally active peptide. Analysis of five INSL3 mutant variants detected in cryptorchid patients showed that P49S substitution renders functionally compromised peptide. Therefore, mutations in INSL3 might contribute to the etiology of cryptorchidism. We have also showed that synthetic insulin-like peptides (INSL4 and INSL6) were unable to activate LGR7 or GREAT/LGR8.     

Suppression of insulin-like3 receptor reveals the role of β-catenin and Notch signaling in gubernaculum development

During male development, the testes move from a high intraabdominal position and descend into the scrotum. The gubernaculum, an inguinoscrotal ligament connecting the testis to the lower abdomen, is believed to play a critical role in this process. The first stage of testicular descent is controlled by insulin like3 hormone (INSL3), produced in testicular Leydig cells. Deletion of Insl3 or its receptor, Rxfp2, in mice causes cryptorchidism. We produced Cre/loxP regulated shRNA transgenic mice targeting RXFP2 expression. We have shown that the transgene was able to reduce Rxfp2 gene expression and thus behaved as a hypomorphic allele of Rxfp2. Variable degrees of uni- and bilateral cryptorchidism was detected in males with the activated shRNA transgene on an Rxfp2+/- background. Conditional suppression of Rxfp2 in the gubernaculum led to cryptorchidism. Gene expression analysis of a mutant cremasteric sac using Illumina microarrays indicated abnormal expression of a significant number of genes in Wnt/β-catenin and Notch pathways. We have demonstrated profound changes in the expression pattern of β-catenin, Notch1, desmin, and androgen receptor (AR), in Rxfp2-/- male embryos, indicating the role of INSL3 in proliferation, differentiation, and survival of specific cellular components of the gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in the gubernaculum. Males with the deletion of β-catenin or Notch1 in the gubernacular ligament demonstrated abnormal development. Our data indicates that β-catenin and Notch pathways are potential targets of INSL3 signaling during gubernacular development.     

Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice

The insulin-like factor 3 (Insl3), a member of the insulin-like hormone family, is exclusively synthesized in gonads. Our recent analysis of Insl3-deficient mice revealed the regulating role of the Insl3 factor on the gubernaculum development during the transabdominal descent of the testis. Here we define the role of the Insl3 factor by histometric analysis of wild-type and Insl3(-/-) ovaries. Ovaries from 40-day-old- and 6-month-old Insl3(-/-) mice as well as from wild-type littermates were serially sectioned. Sections were stained with periodic acid Schiff reaction (PAS) for counting the number of zonae pellucidae which indicated the final stages of follicular atresia. Corpora lutea were also determined. Some sections were processed using either a modified TUNEL method for in situ detection of apoptosis or a lectin labelling technique with Griffonia simplicifolia I agglutinin (GS I) for endothelial cell occurrence. The number of zonae pellucidae was higher in Insl3-deficient ovaries of both ages than in ovaries of wild-type sisters (P < 0.05 for 40-day-old ovaries; P < 0.01 for 6-month-old ovaries). Additionally, the wild-type mice of both ages possessed threefold more corpora lutea than their Insl3 littermates (P < 0.01 for 40-day-old; P < 0.001 for 6-month-old). In general, wild-type corpora lutea looked healthy, showed GS I-positive endothelial cells and no apoptotic cells. Corpora lutea from mutants were rich in regressing GS I luteal cells, and apoptotic cells appeared. We conclude: Follicular atresia and luteolysis are accelerated in ovaries of Insl3-deficient mice probably because of increased apoptosis. The Insl3 function thus appears to rescue endocrine cells from the apoptotic pathway.     

Cryptorchidism in mice with an androgen receptor ablation in gubernaculum testis

Androgens play a critical role in the development of the male reproductive system, including the positioning of the gonads. It is not clear, however, which developmental processes are influenced by androgens and what are the target tissues and cells mediating androgen signaling during testicular descent. Using a Cre-loxP approach, we have produced male mice (GU-ARKO) with conditional inactivation of the androgen receptor (Ar) gene in the gubernacular ligament connecting the epididymis to the caudal abdominal wall. The GU-ARKO males had normal testosterone levels but developed cryptorchidism with the testes located in a suprascrotal position. Although initially subfertile, the GU-ARKO males became sterile with age. We have shown that during development, the mutant gubernaculum failed to undergo eversion, a process giving rise to the processus vaginalis, a peritoneal outpouching inside the scrotum. As a result, the cremasteric sac did not form properly, and the testes remained in the low abdominal position. Abnormal development of the cremaster muscles in the GU-ARKO males suggested the participation of androgens in myogenic differentiation; however, males with conditional AR inactivation in the striated or smooth muscle cells had a normal testicular descent. Gene expression analysis showed that AR deficiency in GU-ARKO males led to the misexpression of genes involved in muscle differentiation, cell signaling, and extracellular space remodeling. We therefore conclude that AR signaling in gubernacular cells is required for gubernaculum eversion and outgrowth. The GU-ARKO mice provide a valuable model of isolated cryptorchidism, one of the most common birth defects in newborn boys.     

A molecular basis for estrogen-induced cryptorchidism

Male sexual differentiation relies upon testicular secretion of the hormones testosterone, Mullerian inhibiting substance, and insulin-3 (Insl3). Insl3 is responsible for testicular descent through virilization and outgrowth of the embryonic gubernaculum. In mouse, prenatal exposure to 17beta-estradiol and the nonsteroidal synthetic estrogen diethylstilbestrol (DES) disturbs the endocrine balance, causing demasculinizing and feminizing effects in the male embryo, including impaired testicular descent (cryptorchidism). In the current study, we show that maternal exposure to estrogens, including 17alpha- and beta-estradiol, as well as DES, specifically down regulates Insl3 expression in embryonic Leydig cells, thereby providing a mechanism for cryptorchidism. These experiments may have implications for the widespread use of estrogenic substances in agriculture and the environment.     

Role of the fetal pituitary in cryptorchidism induced by exogenous maternal oestrogen during pregnancy in mice

A single subcutaneous injection of 5 or 1 mg oestradiol given to pregnant female mice on Day 14 of pregnancy resulted in all male offspring being cryptorchid. Pituitary LH content, testicular weights and structure, seminal vesicle weights and the structure of the reproductive tract as a whole were monitored on the day of birth and at 2, 4, 8 and 14 weeks of age. Apart from an initial significant reduction in pituitary LH at the time of birth, no other marked differences were seen between control and treated animals except that all oestrogen-treated males lacked a gubernaculum and the testes were freely mobile within the abdomen. Hypogonadal (hpg) male mice lacking GnRH are cryptorchid but have a normal gubernaculum and their testes develop and descend normally if treated with gonadotrophins. When the mothers of hpg mice were treated with oestradiol the male offspring lacked a gubernaculum. These results indicate that perturbations of the fetal hypothalamic/pituitary axis play no significant part in oestrogen-induced cryptorchidism in mice.     

Cryptorchidism: an indicator of testicular dysgenesis?

Cryptorchidism is a common ailment of new-born boys, affecting 1–9% of full term boys at birth. Cryptorchidism has been associated with an increased risk of testicular cancer and reduced fertility. Aetiology of cryptorchidism remains obscure in most cases. Familial occurrence suggests a heritable susceptibility to cryptorchidism; however, seasonal variation in the incidence of cryptorchidism suggests that environmental factors also contribute. Testicular descent is characterised by androgen-dependent regression of cranial suspensory ligament and androgen + insulin-like hormone 3 (Insl3)-dependent gubernacular outgrowth. Even though hormonal defects are rarely detected in patients, both hypo-and hypergonadotropic hormonal patterns have been associated with cryptorchidism. Moreover, cryptorchid boys have significantly reduced serum androgen bioactivity at 3 months of age when normal boys have a strong surge of reproductive hormones. Defects in Insl3 action cause cryptorchidism in male mice, and over-expression in female mice causes ovarian descent. Defects in leucine-rich repeat-containing G-protein-coupled receptor 8/G-protein-coupled receptor affecting testis descent (LGR8/GREAT), the receptor for Insl3, manifest the same phenotype as Insl3 knockout mutants. Even though mutations found in Insl3 and LGR8/GREAT genes are not a common cause of cryptorchidism in patients, it remains to be resolved whether low Insl3 levels during development are associated with cryptorchidism. Cryptorchidism may reflect foetal testicular dysgenesis that may later manifest as subfertility or testicular cancer.This work was supported by the Turku University Central Hospital, the Academy of Finland and the European Commission (contracts BMH4-CT96-0314, QLK4-CT1999-01422, QLK4-CT2001-00269 and QLK4-CT2002-00603).     

Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.     

Failure of spermatogenesis in mice lacking connexin43

Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a "Sertoli cell only" phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.     

Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo.Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice.The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.     

Experimental manipulation of sexual differentiation in wallaby pouch young treated with exogenous steroids

We have investigated the effects of androgen or oestrogen treatment of female or male tammar wallabies from the day of birth, when the gonads are histologically undifferentiated, to day 25 of pouch life, when the gonads and the Wolffian and Müllerian ducts have differentiated and the testes have migrated through the inguinal canal. Female tammars treated with testosterone propionate (24-50 mg kg-1 day-1) orally for 25 days had enlarged Wolffian and Müllerian ducts. Mammary and pouch development, however, was indistinguishable from that of control females. The treatment had no apparent effect on ovarian development, or on ovarian position in the abdomen. The phallus of males and females was similar in size, and neither experimental treatment had a significant effect on its size at day 25. Male tammars treated with oestradiol benzoate (1.2-2.5 mg kg-1 day-1) orally for 25 days had gross hypertrophy of the urogenital sinus. Testicular morphology was abnormal; many of the germ cells appeared necrotic, the seminiferous tubules were of reduced diameter, and there were few Leydig cells and increased amounts of fibrous tissue between the tubules. The cortex of these gonads contained some areas which had an ovarian appearance, lacking tubules and containing numerous germ cells. The Müllerian ducts of control males had regressed, but this was prevented by oestrogen treatment, suggesting an inhibition of either Müllerian Inhibiting Substance (MIS) production or its action. Normal testicular migration was inhibited in treated males; the testes remained high in the abdomen, similar in position to the ovaries of control females, whilst control males all had testes in the inguinal region. The gubernaculum and processus vaginalis of control males extended into the scrotum, but in treated males they terminated outside it. Oestrogen treatment had no effect on the size of the scrotum and did not induce mammary or pouch development. These experiments show that marsupials, like eutherians, have a dual hormonal control of Wolffian and Müllerian development. By contrast, the initial development of the mammary glands, pouch, gubernaculum and scrotum does not appear to be under hormonal control and is therefore likely to be autonomous and dependent on genotype.     

Identification of a novel population of adrenal-like cells in the mammalian testis

Steroidogenic cells of the adrenal and gonad are thought to be derived from a common primordium that divides into separate tissues during embryogenesis. In this paper, we show that cells with mixed adrenal and Leydig cell properties are found dispersed in the insterstitium of the embryonic and adult mouse testis. They express the adrenal markers Cyp11b1 and Cyp21 and respond to ACTH. Consistent with these properties, we show that the embryonic testis produces the adrenal steroid corticosterone. These cells also express Cyp17 and respond to hCG stimulation but do not express the Leydig specific marker Insl3 showing that they are a population of steroidogenic cells distinct from Leydig cells. Based on their properties, we refer to these cells as adrenal-like cells of the testis and propose that they are the mouse equivalent of the precursors of human adrenal rests, tumors found primarily in male patients with congenital adrenal hyperplasia. Organ culture studies show that ACTH-responsive cells are present at the gonad/mesonephros border and seem to migrate into the XY but not the XX gonad during development. Consistent with this, using transgenic Cyp11a1 reporter mice, we definitively show that steroidogenic cells can migrate from the mesonephros into the XY gonad. We also show that the region between the mesonephros and the gonad harbors steroidogenic cell precursors that are repressed by the presence of the mesonephros. We propose that this region is the source of the adrenal-like cells that migrate into the testis as it develops and are activated when Leydig cells differentiate. These studies reveal the complex nature of steroidogenic cell differentiation during urogenital development.     

Diethylstilbestrol regulates mouse gubernaculum testis cell proliferation via PLC‐Ca2+‐CREB pathway

Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U‐73122 and then treated with DES. The results demonstrated that U‐73122 impaired DES‐evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES‐induced proliferation of gubernaculum testis cells. Mechanistically, we found that U‐73122 inhibited DES‐induced activation of cAMP‐response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC‐Ca²⁺‐CREB pathway.

Significance of the study

Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC‐Ca²⁺‐CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation.     

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from We/We embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.