Immune and metabolic challenges induce changes in pain sensation and related pathways in the hypothalamus.

The hypothalamic molecular processes participate in the regulation of the neuro-immune-endocrine system, including hormone, metabolite, chemokine circulation, and corresponding physiological and behavioral responses. RNA-sequencing profiles were analyzed to understand the effect of juvenile immune and metabolic distress 100 days after virally elicited maternal immune activation during gestation in pigs. Over 1,300 genes exhibited significant additive or interacting effects of gestational immune activation, juvenile distress, and sex. One-third of these genes presented multiple effects, emphasizing the complex interplay of these factors. Key functional categories enriched among affected genes included sensory perception of pain, steroidogenesis, prolactin, neuropeptide, and inflammatory signaling. These categories underscore the intricate relationship between gestational immune activation during gestation, distress, and the response of hypothalamic pathways to insults. These effects were sex-dependent for many genes, such as Prdm12, Oprd1, Isg20, Prl, Oxt, and Vip. The prevalence of differentially expressed genes annotated to proinflammatory and cell cycle processes suggests potential implications for synaptic plasticity and neuronal survival. The gene profiles affected by immune activation, distress, and sex pointed to the action of transcription factors SHOX2, STAT1, and REST. These findings underscore the importance of considering sex and postnatal challenges when studying causes of neurodevelopmental disorders and highlight the complexity of the "two-hit" hypothesis in understanding their etiology. Our study furthers the understanding of the intricate molecular responses in the hypothalamus to gestational immune activation and subsequent distress, shedding light on the sex-specific effects and the potential long-lasting consequences on pain perception, neuroendocrine regulation, and inflammatory processes.NEW & NOTEWORTHY The interaction of infection during gestation and insults later in life influences the molecular mechanisms in the hypothalamus that participate in pain sensation. The response of the hypothalamic transcriptome varies between sexes and can also affect synapses and immune signals. The findings from this study assist in the identification of agonists or antagonists that can guide pretranslational studies to ameliorate the effects of gestational insults interacting with postnatal challenges on physiological or behavioral disorders.

The effects of short-term and long-term phthalate exposures on ovarian follicle growth dynamics and hormone levels in female mice†.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

Uterine Fibroids: Hiding in Plain Sight.

Uterine fibroids (leiomyomas) are present in >75% of women and can cause serious morbidity. They are by far the leading cause of hysterectomy. Fibroids are a complex mixture of cells that include fibroblasts and smooth muscle cells. Rich in extracellular matrix, they typically arise through somatic mutations, most commonly MED12. Their lack of growth inhibition and their ability to have facets of malignancy yet be histologically and biologically benign provide opportunities to explore basic processes. To date, the mechanisms responsible for growth and development of leiomyomas are an enigma. This review provides an overview of current understanding and future directions for clinical and basic research of fibroids.

Endometriosis leads to central nervous system-wide glial activation in a mouse model of endometriosis.

BACKGROUND: Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied. METHODS: Endometriosis was induced in female mice (45-day-old; n = 6-11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning "Weka trainable segmentation" plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated. RESULTS: We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw. CONCLUSION: We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.

Influenza A virus infection disrupts oligodendrocyte homeostasis and alters the myelin lipidome in the adult mouse.

BACKGROUND: Recent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior. METHODS: Mice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression. RESULTS: Transcriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability. CONCLUSIONS: These findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process.

Basigin is necessary for normal decidualization of human uterine stromal cells.

STUDY QUESTION: Does basigin (BSG) regulate human endometrial stromal cell (HESC) decidualization in vitro? SUMMARY ANSWER: BSG regulates HESCs proliferation and decidualization. WHAT IS KNOWN ALREADY: Studies have shown that in the human endometrium, BSG expression is menstrual-cycle dependent and its expression was significantly lower in uterine endometrium during the luteal phase of women experiencing multiple implantation failures after IVF than in women with normal fertility. STUDY DESIGN, SIZE, DURATION: We utilized a telomerase-immortalized HESCs in an in vitro cell culture model system to investigate whether BSG regulates decidualization of stromal cells. Further, we used microarray analysis to identify changes in the gene expression profile of HESCs treated with BSG small interfering RNA (siRNA). All experiments were repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of BSG knockdown (using siRNA) on HESC proliferation was determined by counting cell number and by tritiated thymidine incorporation assays. The effect of BSG on decidualization of HESCs was determined by RT-qPCR for the decidualization markers insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL). Immunoblotting was used to determine the effect of BSG siRNA on the expression of MMP-2,3. Microarray analysis was used to identify BSG-regulated genes in HESCs at Day 6 of decidualization. Functional and pathway enrichment analyses were then carried out on the differentially expressed genes (DEGs). The STRING online database was used to analyze protein-protein interaction (PPI) between DEG-encoded proteins, and CytoScape software was used to visualize the interaction. MCODE and CytoHubba were used to construct functional modules and screen hub genes separately. Several BSG-regulated genes identified in the microarray analysis were confirmed by qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: Knockdown of BSG expression in cultured stromal cells by siRNA significantly (P < 0.05) inhibited HESC proliferation, disrupted cell decidualization and down-regulated MMP-2 and MMP-3 expression. Microarray analysis identified 721 genes that were down-regulated, and 484 genes up-regulated with P < 0.05 in BSG siRNA treated HESCs. GO term enrichment analysis showed that the DEGs were significantly enriched in cell communication, signaling transduction and regulation, response to stimulus, cell adhesion, anatomical structure morphogenesis, extracellular matrix organization, as well as other functional pathways. KEGG pathway analysis identified upregulated gene enriched in pathways such as the MAPK signaling pathway, colorectal cancer, melanoma and axon guidance. In contrast, downregulated genes were mainly enriched in pathways including ECM-receptor interaction, PI3K-Akt signaling pathway, pathways in cancer, antigen processing, type I diabetes mellitus and focal adhesion. The top 10 hub nodes were identified using 12 methods analyses. The hub genes that showed up in two methods were screened out. Among these genes, upregulated genes included EGFR, HSP90AA1, CCND1, PXN, PRKACB, MGAT4A, EVA1A, LGALS1, STC2, HSPA4; downregulated genes included WNT4/5, FOXO1, CDK1, PIK3R1, IGF1, JAK2, LAMB1, ITGAV, HGF, MXRA8, TMEM132A, UBE2C, QSOX1, ERBB2, GNB4, HSP90B1, LAMB2, LAMC1 and ITGA1. Hub genes and module genes involved in the top three modules of PPI analysis were analyzed through the string database. Analysis showed that hub and module genes were related mainly to the WNT signaling pathway, PI3K-AKT signaling pathway and pathways in cancer. LARGE SCALE DATA: The microarray data set generated in this study has been published online at databank.illinois.edu. LIMITATIONS, REASONS FOR CAUTION: Most of the findings were obtained using an in vitro cell culture system that may not necessarily reflect in vivo functions. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that BSG plays a vital role in decidualization and that downregulation of BSG in the uterine endometrium may be associated with infertility in women. The identified hub genes and pathways increase our understanding of the genetic etiology and molecular mechanisms underlying the regulation of decidualization by BSG. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the NIH U54 HD40093 (R.A.N.). The authors have no competing interests to declare.

Virilizing doses of testosterone decrease circulating insulin levels and differentially regulate insulin signaling in liver and adipose tissue of females.

Transgender men undergoing hormone therapy are at risk for insulin resistance. However, how virilizing testosterone therapy affects serum insulin and peripheral insulin sensitivity in transgender men is unknown. This study assessed the effect of acute, virilizing testosterone on serum insulin concentrations and insulin signaling in liver, skeletal muscle, and white adipose tissue (WAT) of female pigs as a translational model for transgender men. Females received three doses of intramuscular testosterone cypionate (TEST females; 50 mg/day/pig) or corn oil (control) spaced 6 days apart starting on the day of estrus (D0). Fasting blood was collected on D0, D3, D5, D11, and D13, and females were euthanized on D13. On D13, TEST females had virilizing concentrations of serum testosterone with normal concentrations of serum estradiol. Virilizing serum testosterone concentrations (D13) were associated with decreased serum insulin and C-peptide concentrations. Blood glucose and serum glycerol concentrations were not altered by testosterone. Virilizing concentrations of testosterone downregulated AR and ESR1 in subcutaneous (sc) WAT and upregulated transcript levels of insulin-signaling pathway components in WAT and liver. At the protein level, virilizing testosterone concentrations were associated with increased PI3K 110α in liver and increased insulin receptor (INSR) and phospho(Ser256)-FOXO1 in visceral (v) WAT but decreased phospho(Ser473)-AKT in vWAT and scWAT. These results suggest that acute exposure to virilizing concentrations of testosterone suppresses circulating insulin levels and results in increased abundance of proteins in the insulin-signaling pathway in liver and altered phosphorylation of key proteins in control of insulin sensitivity in WAT.NEW & NOTEWORTHY Acute virilizing doses of testosterone administered to females suppress circulating insulin levels, upregulate components of the insulin-signaling pathway in liver, and suppress insulin signaling in white adipose tissue. These results suggest that insulin resistance in transgender men may be due to suppression of the insulin-signaling pathway and decreased insulin sensitivity in white adipose tissue.

The effects of the phthalate DiNP on reproduction†.

Di-isononyl phthalate (DiNP) is a high molecular weight, general purpose, plasticizer used primarily in the manufacture of polymers and consumer products. It can be metabolized rapidly and does not bioaccumulate. The primary metabolite of DiNP is monoisononyl-phthalate (MiNP) and the secondary metabolites include three oxidative derivatives of DiNP, which have been identified mainly in urine: mono-oxoisononyl phthalate (MOINP or oxo-MiNP), mono-carboxyisooctyl phthalate (MCIOP, MCOP or cx-MiNP), and mono-hydroxyisononyl phthalate (MHINP or OH-MiNP). The secondary metabolites are very sensitive biomarkers of DiNP exposure while primary metabolites are not. As the usage of DiNP worldwide increases, studies evaluating its potential reproductive toxicity are becoming more prevalent in the literature. In studies on female animals, the researchers found that the exposure to DiNP appears to induce negative effects on ovarian function and fertility in animal models. Whether or not DiNP has direct effects on the uterus is still controversial, and the effects on human reproduction require much more research. Studies on males indicate that DiNP exposure has disruptive effects on male reproduction and fertility. Occupational studies also indicate that the exposure to DiNP might induce negative effects on male reproduction, but larger cohort studies are needed to confirm this. This review presents an overview of the literature regarding the reproductive effects of exposure to DiNP.

Loss of basigin expression in uterine cells leads to subfertility in female mice†.

Basigin (BSG) is a transmembrane glycoprotein involved in cell proliferation, angiogenesis, and tissue remodeling. BSG has been shown to be essential for male and female reproduction although little is known about its role in normal uterine function. To study the potential function of BSG in the female reproductive tract, we generated mice with conditional knockout of Bsg in uterine cells using progesterone receptor-Cre and hypothesized that BSG is required for normal pregnancy in mice. Fertility study data showed that the conditional knockout mice had significantly reduced fertility compared to controls. Ovarian function of the conditional knockout mice appeared normal with no difference in the number of superovulated oocytes collected or in serum progesterone levels between the conditional knockout and the control mice. Uterine tissues collected at various times of gestation showed increased abnormalities in implantation, decidualization, placentation, and parturition in the conditional knockout mice. Uterine cross sections on Day 5 of pregnancy showed implantation failure and abnormal uterine epithelial differentiation in a large proportion of the conditional knockout mice. There was a compromised decidual response to artificial decidualization stimuli and decreased mRNA and protein levels for decidualization genes in the uteri of the conditional knockout mice. We also observed altered protein expression of monocarboxylate transporter 1 (MCT1), as well as impaired angiogenesis in the conditional knockout uteri compared to the controls. These results support that BSG is required for successful pregnancy through its functions in implantation and decidualization.

Role of monocarboxylate transporters in regulating metabolic homeostasis in the outer retina: Insight gained from cell-specific Bsg deletion.

The neural retina metabolizes glucose through aerobic glycolysis generating large amounts of lactate. Lactate flux into and out of cells is regulated by proton-coupled monocarboxylate transporters (MCTs), which are encoded by members of the Slc16a family. MCT1, MCT3, and MCT4 are expressed in the retina and require association with the accessory protein basigin, encoded by Bsg, for maturation and trafficking to the plasma membrane. Bsg-/- mice have severely reduced electroretinograms (ERGs) and progressive photoreceptor degeneration, which is presumed to be driven by metabolic dysfunction resulting from loss of MCTs. To understand the basis of the Bsg-/- phenotype, we generated mice with conditional deletion of Bsg in rods (RodΔBsg), cones (Cone∆Bsg), or retinal pigment epithelial cells (RPEΔBsg). RodΔBsg mice showed a progressive loss of photoreceptors, while ConeΔBsg mice did not display a degenerative phenotype. The RPEΔBsg mice developed a distinct phenotype characterized by severely reduced ERG responses as early as 4 weeks of age. The loss of lactate transporters from the RPE most closely resembled the phenotype of the Bsg-/- mouse, suggesting that the regulation of lactate levels in the RPE and the subretinal space is essential for the viability and function of photoreceptors.

Gonad development and reproductive hormones of invasive silver carp (Hypophthalmichthys molitrix) in the Illinois River.

Reproduction is a major component of an animal's life history strategy. Species with plasticity in their reproductive biology are likely to be successful as an invasive species, as they can adapt their reproductive effort during various phases of a biological invasion. Silver carp (Hypophthalmicthys molitrix), an invasive cyprinid in North America, display wide variation in reproductive strategies across both their native and introduced ranges, though the specifics of silver carp reproduction in the Illinois River have not been established. We assessed reproductive status using histological and endocrinological methods in silver carp between April and October 2018, with additional histological data from August to October 2017. Here, we show that female silver carp are batch spawners with asynchronous, indeterminate oocyte recruitment, while male silver carp utilize a determinate pattern of spermatogenesis which ceases in the early summer. High plasma testosterone levels in females could be responsible for regulating oocyte development. Our results suggest that silver carp have high spawning activity in the early summer (May-June), but outside of the peak spawning period, female silver carp can maintain spawning-capable status by adjusting rates of gametogenesis and atresia in response to environmental conditions, while males regress their gonads as early as July. The results of this study are compared to reports of silver carp reproduction in other North American rivers as well as in Asia.

The role of basigin in reproduction.

Basigin is a highly glycosylated transmembrane protein that was originally identified as a product of tumor cells. Basigin is a potent inducer of matrix metalloproteinases (MMPs) and angiogenic factors such as vascular endothelial growth factor (VEGF). Basigin is also a chaperone protein for specific metabolite transporters in the plasma cell membrane such as the monocarboxylate transporters and is an important regulator of cell metabolism. Studies in reproductive model systems have demonstrated that basigin is expressed in the testis, ovary, uterus and placenta and is necessary for normal fertility in both males and females. Overexpression of basigin is associated with reproductive diseases including uterine leiomyomas and endometriosis. This review presents an overview of the literature regarding the physiological role of basigin in reproductive tissues and the mechanistic pathways involved in its actions.

Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model.

STUDY QUESTION: Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model? SUMMARY ANSWER: HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis. WHAT IS KNOWN ALREADY: Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas. STUDY DESIGN, SIZE, DURATION: Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P < 0.05) reduction in tumor volume. The HF-induced volume reduction was accompanied by increased apoptosis and decreased cell proliferation. In contrast, there was no significant change in the collagen content either at the transcript or protein level between UL xenografts in control and HF groups. HF treatment did not change the expression level of ovarian steroid hormone receptors. No adverse pathological effects were observed in other tissues from mice undergoing treatment at these doses. LIMITATIONS, REASONS FOR CAUTION: While this study did test the effects of HF on human leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery. STUDY FUNDING/COMPETING INTERESTS: This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests.

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway.

Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase-protein kinase B/AKT-mammalian target of rapamycin (PI3K/AKT-mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K-AKT-mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids.

Basigin null mutant male mice are sterile and exhibit impaired interactions between germ cells and Sertoli cells.

Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa.