Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors

Follicle stimulating hormone (FSH) and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action we have crossed hypogonadal (hpg) mice which lack gonadotrophins with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers assessed. In untreated hpg and hpg.SCARKO mice germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice FSH increased numbers of spermatogonia and spermatocytes and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs

Novas perspectivas no diagnóstico do hipogonadismo pediátrico masculino: a importância do AMH como marcador de células de Sertoli

Sertoli cells are the most active cell population in the testis during infancy and childhood. In these periods of life, hypogonadism can only be evidenced without stimulation tests, if Sertoli cell function is assessed. AMH is a useful marker of prepubertal Sertoli cell activity and number. Serum AMH is high from fetal life until mid-puberty. Testicular AMH production increases in response to FSH and is potently inhibited by androgens. Serum AMH is undetectable in anorchidic patients. In primary or central hypogonadism affecting the whole gonad and established in fetal life or childhood, serum AMH is low. Conversely, when hypogonadism affects only Leydig cells (e.g. LHb mutations, LH/CG receptor or steroidogenic enzyme defects), serum AMH is normal or high. In pubertal males with central hypogonadism, AMH is low for Tanner stage (reflecting lack of FSH stimulus), but high for the age (indicating lack of testosterone inhibitory effect). Treatment with FSH provokes an increase in serum AMH, whereas hCG administration increases testosterone levels, which downregulate AMH. In conclusion, assessment of serum AMH is helpful to evaluate gonadal function, without the need for stimulation tests, and guides etiological diagnosis of pediatric male hypogonadism. Furthermore, serum AMH is an excellent marker of FSH and androgen action on the testis.b mutations, LH/CG receptor or steroidogenic enzyme defects), serum AMH is normal or high. In pubertal males with central hypogonadism, AMH is low for Tanner stage (reflecting lack of FSH stimulus), but high for the age (indicating lack of testosterone inhibitory effect). Treatment with FSH provokes an increase in serum AMH, whereas hCG administration increases testosterone levels, which downregulate AMH. In conclusion, assessment of serum AMH is helpful to evaluate gonadal function, without the need for stimulation tests, and guides etiological diagnosis of pediatric male hypogonadism. Furthermore, serum AMH is an excellent marker of FSH and androgen action on the testis.As células de Sertoli são a população de células mais ativa nos testículos durante a primeira e segunda infância. Neste período, o hipogonadismo só pode ser evidenciado sem o uso de testes estimulatórios se a função das células de Sertoli for avaliada. O AMH é um marcador útil do número e da atividade das células de Sertoli no período pré-puberal. A concentração sérica de AMH é alta da metade da vida fetal até a metade da puberdade. A produção de AMH pelos testículos aumenta em resposta ao FSH e é potencialmente inibida por androgênios. O AMH sérico não é detectável em pacientes anorquídicos. No hipogonadismo central ou primário afetando a gônada inteira, ou estabelecido na vida fetal ou infância, a concentração de AMH sérica é baixa. Por outro lado, quando o hipogonadismo afeta apenas as células de Leydig (por exemplo, nas mutações, LHb, defeitos do receptor de LH/CG ou das enzimas esteroidogênicas), a concentração de AMH sérico é normal ou alta. Em meninos púberes com hipogonadismo central, a concentração de AMH é baixa para o estágio na escala de Tanner (refletindo a falta de estímulo pelo FSH), mas alta para a idade (indicando a falta do efeito inibidor da testosterona). O tratamento com FSH provoca um aumento do AMH sérico, enquanto a administração de hCG aumenta os níveis de testosterona, que fazem a downregulation do AMH. Em conclusão, a concentração sérica de AMH é útil na avaliação da função gonadal, excluindo a necessidade de testes estimulatórios, e direciona o diagnóstico etiológico do hipogonadismo pediátrico masculino. Além disso, o AMH sérico é um marcador excelente da ação do FSH e dos androgênios nos testículosFil: Grinspon, Romina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rey, Rodolfo Alberto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología; Argentin

Development and application of a bioassay for follicle-stimulating hormone : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of vital reproductive processes, such as gametogenesis, follicular development and ovulation. Produced in the anterior pituitary, FSH is a glycoprotein hormone that exists as a family of isohormones. Follicle-stimulating hormone concentrations have traditionally been measured by radioimmunoassay (RIA). However, results generated using RIA are a determination of the immunological activity of FSH. The potential of FSH to generate a biological response cannot be measured by RIA. Therefore, the identification of physiologically significant differences in the activity of these isoforms requires the use of assay systems that can differentiate between the biological activity of the FSH isoforms. Commonly used assays for measuring the biological activity of FSH are based on the measurement of aromatase activity in cultured rat Sertoli cells following stimulation with FSH. However, these assays have an inherently high ethical cost involved due to the use of primary tissue culture. In addition, the variation in these assays associated with differences between animals is difficult to eliminate. Recently a bioassay for human FSH has been described based on FSH stimulation of cyclic AMP production by a Chinese hamster ovary (CHO) cell line stably expressing the human FSH receptor (FSH-R). The purpose of this study was to evaluate the potential usefulness of this CHO FSH-R cell line expressing the human receptor for FSH to be used as a bioassay to measure the biological activity of ovine FSH. The receptor cell line bioassay described in this study is based on the ability of FSH to stimulate cAMP production by cultured CHO FSH-R cells. Optimisation of the culture system to enable the bioactivity of ovine FSH to be measured by bioassay was undertaken. This involved optimising the density of cultured cells, the time in culture and time exposed to FSH and the most suitable dose range for FSH. The influence of matrix effects, such as those exerted by serum was also investigated. The specificity of the assay towards FSH was also determined as was the sensitivity, accuracy and precision of the assay. No stimulation of cAMP production was seen in CHO FSH-R cells following treatment with α-FSH, β-FSH, LH, TSH, GH, prolactin or vasopressin at concentrations up to 10 μg/ml. Although the methodology used differed slightly depending on the presence or absence of serum, all assayed were performed using the following methods and materials. Freshly thawed FSH-R cells were bulked up in culture, and aliquots of 1 x 105 to 5 x 105 cells/well dispensed into 48 well culture dishes and incubated overnight at 37°C. The assay culture media was then replaced with 0.25 ml fresh media (α-MEM + 0.1% BSA + 0.25 mM 3-isobutyl-1-methyl-xanthine) containing varying doses of NIH-FSH-RP2 (RP2) FSH preparations or FSH containing samples, and the cells incubated for 4 hours at 37°C. The assay culture media was then removed and stored frozen at -20°C until assayed for cyclic adenosine monophosphate (cAMP) by RIA. Once optimal assay conditions were determined, the CHO FSH-R cell bioassay was used to measure FSH concentrations in ovine serum, pituitary extracts and medium from cultures of ovine pituitary cells. It was found that the concentrations of FSH in serum from intact sheep was close to the detection limit of the assay. Thus, while FSH concentrations could be measured in serum from some sheep, other animals had concentrations that were too low to be accurately measured by the bioassay in its present form. The assay was, however, well suited to measuring FSH concentrations in serum from sheep that had elevated concentrations of FSH. In one study, FSH concentrations measured by the bioassay were compared to those measured by RIA in sheep that had been ovariectomised and then hypophysectomised. It was found that the profile of FSH concentrations following hypophysectomy was similar whether measured by RIA or by bioassay (R2=0.7513), though absolute concentrations sometimes differed. This suggested that the immunoassay and bioassay were not always measuring the same characteristics of FSH. The assay was also used to measure FSH concentrations in samples of ovine hypophyseal venous blood. However, the results obtained for these samples indicated a poor correlation between FSH concentrations obtained by bioassay and RIA. Levels of bioactive FSH in hypophyseal venous blood fluctuated markedly and were up to 10-fold higher than the associated RIA concentrations. The CHO-cell bioassay was also found to be very suitable for measuring pituitary concentrations of FSH. In one study, pituitary extracts underwent chromatography and the separated isoforms of FSH were analysed by bioassay and RIA. Again, there was excellent correlation (R2=0.9328) between the concentrations of FSH measured both assay types. However, some differences were apparent suggesting a discrepancy in the biological and immunological characteristics of different FSH isoforms. The bioassay was also used to measure FSH concentrations in media from pituitary cells in tissue culture where serially diluted samples displayed good parallelism with the RP2 FSH standard curve. Results of this study demonstrate that the CHO FSH-R cell bioassay is suitable for measuring the biological activity of ovine FSH in a variety of biological fluids. The use of a permanent cell line eliminates the high ethical cost associated with primary tissue culture that other bioassay systems have. The inherent variation associated with culture systems utilising tissue from different sources is also avoided. The sensitivity of the bioassay is suitable for measuring FSH in surgically altered sheep or hypophyseal blood concentrations where FSH levels are generally higher than those in the peripheral circulation. In addition to blood samples, the bioassay is also excellent for monitoring FSH activity in pituitary extracts and in media from tissue culture. However, the sensitivity of the bioassay currently does not always allow measurement of bioactive FSH concentrations in serum samples with low FSH levels. In summary, the CHO FSH-R cell bioassay described in this study offers a useful alternative to RIA and other bioassays for monitoring the biological activity of ovine FSH and its isoforms in various biological fluids. It is concluded that this convenient and robust bioassay may have considerable application in future investigations of ovine FSH bioactivity

Ovarian reserve and anti-Mullerian hormone (AMH) in mothers of dizygotic twins

This study aimed to explore if natural dizygotic (DZ) twinning is associated with earlier menopause and lower anti-Mullerian hormone (AMH) values. We investigated if advanced biological reproductive aging, which can be responsible for the multiple follicle growth in familial twinning, is similar to mechanisms that occur in normal ovarian aging, reflected by earlier menopause in mothers of DZ twins and lower levels of AMH. A total of 16 mothers of DZ twins enrolled with the Netherlands Twin Register (average age at first assessment: 35.9 +/- 3.0 years) and 14 control mothers (35.1 +/- 3 years) took part in a prospective study. Fifteen years after entry into the study, which included follicle-stimulating hormone (FSH) assessment, AMH was measured in stored serum samples and menopause status was evaluated. Average AMH levels were not significantly different between DZ twin mothers and controls (2.1 +/- 2.4 mu g/L vs. 1.9 +/- 1.9 mu g/L). Among the 16 mothers of twins, 7 had an elevated (FSH) value over 10 U/L at first assessment. Their AMH levels were lower than the nine twin mothers with normal FSH values: 0.6 +/- 0.4 versus 3.4 +/- 2.6 mu g/L (p = .01). Of the mothers of twins, eight mothers had entered menopause at the second assessment compared with only one control mother (p = .07). Thus, slightly more DZ mothers were in menopause than the control mothers, although this difference was not significant. The subgroup of DZ twin mothers who had an increased FSH concentration 15 years ago had a limited ovarian reserve as reflected by lower AMH levels. These data indicate that advanced ovarian aging can be a feature in familial DZ twinning, particularly with elevated early follicular phase FSH

Ferromagnet-Superconductor Hybrids

A new class of phenomena discussed in this review is based on interaction between spatially separated, but closely located ferromagnets and superconductors. They are called Ferromagnet-Superconductor Hybrids (FSH). These systems include coupled smooth and textured Ferromagnetic and Superconducting films, magnetic dots, wires etc.The interaction may be provided by the magnetic flux from magnetic textures and supercurrents. The magnetic flux from magnetic textures or topological defects can pin vortices or create them, changing drastically the properties of the superconductor. On the other hand, the magnetic field from supercurrents (vortices) strongly interacts with the magnetic subsystem leading to formation of coupled magnetic-superconducting topological defects. We discuss possible experimental realization of the FSH. The presence of ferromagnetic layer can change dramatically the properties of the superconducting film due to proximity effect. We discuss experimental and theoretical studies of the proximity effect in the FSH including transition temperature and order parameter oscillations and triplet superconductivity.Comment: 68 pages, submited to Advances of Physic

The effects of uterine artery embolisation and surgical treatment on ovarian function in women with uterine fibroids

Objective The aim of this study was to evaluate and compare both ovarian function and menstrual characteristics following uterine artery embolisation (UAE) and surgery. Design Subgroup of women from a randomised controlled trial. Setting Gynaecology and radiology units in Scotland, UK. Population Ninety-six women from the randomised controlled trial comparing embolisation with surgery as a treatment for fibroids (REST), which recruited 157 patients (106 UAE; 51 surgery). Methods Seventy-three women undergoing UAE and 23 women undergoing surgery (with ovarian conservation) had serum follicle-stimulating hormone (FSH) measurements taken on day 3 of the menstrual cycle prior to treatment, and at 6 and 12 months post-treatment. Data on menstrual cycle characteristics was also collected. Main outcome measures Ovarian failure, as defined by an FSH level of > 40 iu/l, and change in duration of menses and length of menstrual cycle. Results There was no significant difference in the rate of ovarian failure at 12 months between UAE (11%) and surgical patients (18%) (P = 0.44). This finding was not influenced by age. The mean duration of menstrual flow decreased significantly, from baseline to 12 months, by 1.7 days (SD 3.8), (95% CI 0.8-2.6). There was no statistically significant change in mean cycle length at 12 months (0.7 days [SD 4.9]; 95% CI [-0.5, 1.9]). Conclusions There is no evidence for UAE accelerating a deterioration in ovarian function at 1 year, when compared with surgery. UAE is associated with a decrease in the duration of menstrual flow at 1 yea

Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells: data from mutant and genetically modified mice

Development and maintenance of the male phenotype and establishment of fertility are all dependent upon the activity of the Sertoli cells and Leydig cells of the testis. This review examines the regulation and function of these cell during fetal and post-natal development. Fetal Leydig cells are sensitive to both luteinising hormone (LH) and adrenocorticotrophic hormone (ACTH) but Leydig cell function appears normal in fetal mice lacking both hormones or their receptors. Post-natally, the Sertoli cells and Leydig cells are reliant upon the pituitary gonadotrophins. Leydig cells are critically dependent on LH but follicle-stimulating hormone (FSH), presumably acting through the Sertoli cell, can also affect Leydig cell function. Testosterone secreted by the Leydig cells acts with FSH to stimulate Sertoli cell activity and spermatogenesis. Study of animals lacking FSH-receptors and androgen-receptors shows that both hormones can act to maintain the meiotic germ cell population but that androgens are critical for completion of meiosis

Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice.

cFOS is a pleiotropic transcription factor, which binds to the AP1 site in the promoter of target genes. In the pituitary gonadotropes, cFOS mediates induction of FSHβ and GnRH receptor genes. Herein, we analyzed reproductive function in the cFOS-deficient mice to determine its role in vivo. In the pituitary cFOS is necessary for gonadotropin subunit expression, while TSHβ is unaffected. Additionally, cFOS null animals have the same sex-steroid levels, although gametogenesis is impeded. In the brain, cFOS is not necessary for GnRH neuronal migration, axon targeting, cell number, or mRNA levels. Conversely, cFOS nulls, particularly females, have decreased Kiss1 neuron numbers and lower Kiss1 mRNA levels. Collectively, our novel findings suggest that cFOS plays a cell-specific role at multiple levels of the hypothalamic-pituitary-gonadal axis, affecting gonadotropes but not thyrotropes in the pituitary, and kisspeptin neurons but not GnRH neurons in the hypothalamus, thereby contributing to the overall control of reproduction

Linearizing nonlinear optics

In the framework of linear optics, light fields do not interact with each other in a medium. Yet, when their field amplitude becomes comparable to the electron binding energies of matter, the nonlinear motion of these electrons emits new dipole radiation whose amplitude, frequency and phase differ from the incoming fields. Such high fields are typically achieved with ultra-short, femtosecond (1fs = 10-15 sec.) laser pulses containing very broad frequency spectra. Here, the matter not only couples incoming and outgoing fields but also causes different spectral components to interact and mix through a convolution process. In this contribution, we describe how frequency domain nonlinear optics overcomes the shortcomings arising from this convolution in conventional time domain nonlinear optics1. We generate light fields with previously inaccessible properties because the uncontrolled coupling of amplitudes and phases is turned off. For example, arbitrary phase functions are transferred linearly to the second harmonic frequency while maintaining the exact shape of the input power spectrum squared. This nonlinear control over output amplitudes and phases opens up new avenues for applications based on manipulation of coherent light fields. One could investigate c.f. the effect of tailored nonlinear perturbations on the evolution of discrete eigenmodes in Anderson localization2. Our approach might also open a new chapter for controlling electronic and vibrational couplings in 2D-spectroscopy3 by the geometrical optical arrangement