Murine sex chromosome aneuploidies (SCAs) provide powerful models for charting sex chromosome influences on mammalian brain development. Here, building on prior work in X-monosomic (XO) mice, we use spatially non-biased high-resolution imaging to compare and contrast neuroanatomical alterations in XXY and XO mice relative to their wild-type XX and XY littermates. First, we show that carriage of a supernumerary X chromosome in XXY males (1) does not prevent normative volumetric masculinization of the bed nucleus of the stria terminalis (BNST) and medial amygdala, but (2) causes distributed anatomical alterations relative to XY males, which show a statistically unexpected tendency to be co-localized with and reciprocal to XO-XX differences in anatomy. These overlaps identify the lateral septum, BNST, ventral group thalamic nuclei and periaqueductal gray matter as regions with replicable sensitivity to X chromosome dose across two SCAs. We then harness anatomical variation across all four karyotype groups in our study--XO, XX, XY and XXY--to create an agnostic data-driven segmentation of the mouse brain into five distributed clusters which (1) recover fundamental properties of brain organization with high spatial precision, (2) define two previously uncharacterized systems of relative volume excess in females vs. males ("forebrain cholinergic" and "cerebelo-pontine-thalamo-cortical"), and (3) adopt stereotyped spatial motifs which delineate ordered gradients of sex chromosome and gonadal influences on volumetric brain development. Taken together, these data provide a new framework for the study of sexually dimorphic influences on brain development in health and disrupted brain development in SCA.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines.

Objective

To determine whether a user-controlled sperm concentration test compared with standard semen analysis can effectively monitor spermatogenesis suppression for male contraception.

Design

Single center, prospective sub study of the ongoing clinical trial: "Study of daily application of Nestorone and testosterone combination gel for male contraception."

Setting

Research institute at an academic medical center.

Participant(s)

Couples participating in the male contraceptive clinical trial.

Interventions

None.

Main outcome measure(s)

The ability by participants to monitor sperm suppression to a threshold compatible with contraceptive efficacy utilizing a user-controlled test verified by sperm concentration determined by standard laboratory methods.

Result(s)

Thirty-eight men participating in a hormonal male contraceptive clinical trial provided multiple samples during spermatogenesis suppression for this substudy. Participants, employing a user-controlled test, correctly identified the absence of sperm (a negative test) in 100% of their laboratory-confirmed azoospermic samples (n = 122). Participants also identified 100% of samples (n = 73) with sperm >0.2 million/mL as positive. Sperm counts between 0.01 and 0.2 million/mL were identified as negative in 96% of samples. Trial participants noted the overall ease of using the test with respect to sample preparation, test timing, and result interpretation, and that they could accurately use this test at home without difficulty.

Conclusion(s)

Participants undergoing spermatogenesis suppression in a hormonal male contraceptive trial performed user-controlled test to determine whether their semen sperm concentration was ≤ or >0.2 million/mL. Compared with standard semen analyses, participants correctly identified 100% of samples with sperm counts >0.2 million/mL as positive (Sensitivity 100%). A positive result when the couple is using a male contraceptive method triggers the need for semen analysis by a laboratory while the couple uses another method of contraception. Participants correctly diagnosed samples ≤0.2 million sperm/mL as negative in 99% of samples (specificity 99%). A negative result indicates a sperm concentration ≤0.2 million/mL, well below the threshold of ≤1 million/mL offering contraceptive efficacy demonstrated by prior studies. At-home sperm concentration test would minimize the need for users to return to the clinic to monitor suppression of spermatogenesis, decreasing cost and burden of male contraception trials and increasing practicality of the method.

Clinical trial registration number

NCT: 03452111.

Subfertility is a major concern of long-term cancer survivors at the reproductive age. We have previously demonstrated that a potent humanin analogue, HNG, protected chemotherapy-induced apoptosis in germ cells but not cancer cells in a metastatic melanoma allograft model. In this study, we utilized severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma to study the effect of HNG in Temozolomide (TMZ) induced male germ cell apoptosis and white blood cell (WBC) suppression. Human medulloblastoma DAOY cells were injected subcutaneously into the right flank of male SCID mice. Three weeks later, groups of tumor-bearing mice received one of the following treatments: vehicle, HNG, TMZ, or TMZ + HNG. 24 h after last injection, the tumors weights, complete blood counts, liver and spleen weights, male germ cell apoptosis was assessed. HNG did not affect TMZ's significant anti-tumor action. HNG significantly prevented TMZ-induced germ cell apoptosis and attenuated the suppressed total WBC and granulocyte counts in SCID mice with or without TMZ treatment. HNG also attenuated TMZ-induced body weight loss and decrease of spleen and liver weights. In conclusion, HNG ameliorated TMZ-induced germ cell apoptosis; WBC and granulocytes loss; and decreased body/organ weights without compromising the TMZ's anti-cancer action on medulloblastoma xenografts in SCID mice.

Klinefelter syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (usually XXY), and spermatogonial stem cell (SSC) loss in their early life. Affecting 1 out of every 650 males born, KS is the most common genetic cause of male infertility, and new fertility preservation strategies are critically important for these patients. In this study, testes from 41, XXY prepubertal (3-day-old) mice were frozen-thawed. Isolated testicular cells were cultured and characterized by qPCR, digital PCR, and flow cytometry analyses. We demonstrated that SSCs survived and were able to be propagated with testicular somatic cells in culture for up to 120 days. DNA fluorescent in situ hybridization (FISH) showed the presence of XXY spermatogonia at the beginning of the culture and a variety of propagated XY, XX, and XXY spermatogonia at the end of the culture. These data provide the first evidence that an extra sex chromosome was lost during innate SSC culture, a crucial finding in treating KS patients for preserving and propagating SSCs for future sperm production, either in vitro or in vivo. This in vitro propagation system can be translated to clinical fertility preservation for KS patients.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16⁺ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA^-/CD9⁺/CD49f⁺ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Humanin is a member of a new family of peptides that are encoded by short open reading frames within the mitochondrial genome. It is conserved in animals and is both neuroprotective and cytoprotective. Here we report that in C. elegans the overexpression of humanin is sufficient to increase lifespan, dependent on daf-16/Foxo. Humanin transgenic mice have many phenotypes that overlap with the worm phenotypes and, similar to exogenous humanin treatment, have increased protection against toxic insults. Treating middle-aged mice twice weekly with the potent humanin analogue HNG, humanin improves metabolic healthspan parameters and reduces inflammatory markers. In multiple species, humanin levels generally decline with age, but here we show that levels are surprisingly stable in the naked mole-rat, a model of negligible senescence. Furthermore, in children of centenarians, who are more likely to become centenarians themselves, circulating humanin levels are much greater than age-matched control subjects. Further linking humanin to healthspan, we observe that humanin levels are decreased in human diseases such as Alzheimer's disease and MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes). Together, these studies are the first to demonstrate that humanin is linked to improved healthspan and increased lifespan.