Katja J. Teerds

Functional relationship between obesity and male reproduction: from humans to animal models

BACKGROUND The increase in the incidence of obesity has a substantial societal health impact. Contrasting reports have been published on whether overweight and obesity affect male fertility. To clarify this, we have reviewed published data on the relation between overweight/obesity, semen parameters, endocrine status and human male fertility. Subsequently, we have used results obtained in animal models of obesity to explain the human data. METHODS Pubmed, Scopus, Web of Science and Google Scholar databases were searched between September 2009 and October 2010 for a comprehensive publication record. Available studies on adult human males were examined. The included animal studies examined obesity and fertility, and focused on leptin, leptin receptor signaling, kisspeptins and/or NPY. RESULTS Most overweight/obese men do not experience significant fertility problems, despite the presence of reduced testosterone alongside normal gonadotrophin levels. Only a subgroup of subjects suffers from hypogonadotropic hypogonadism. Animal models offer several explanations and show that reduced leptin signaling leads to reduced GnRH neuronal activity. This may be due to decreased hypothalamic Kiss1 expression, a potent regulator of GnRH/LH/FSH release. As the Kiss1 neurons express leptin receptors, the Kiss1 system may participate in transmitting metabolic information to the GnRH neurons, thus providing a bridge between metabolic regulation and fertility. CONCLUSIONS Infertility in overweight/obese males may be explained by leptin insensitivity. This implies a possible role for the KISS1 system in human obesity-related male infertility. If substantiated, it will pave the way for methods to restore fertility in these subjects.

Propagation of bovine spermatogonial stem cells in vitro

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

The role of luteinizing hormone in the pathogenesis of hyperadrenocorticism in neutered ferrets

Four studies were performed to test the hypothesis that gonadotrophic hormones, and particularly luteinizing hormone (LH) play a role in the pathogenesis of ferrets: (I) adrenal glands of ferrets with hyperadrenocorticism were studied immunohistochemically to detect LH-receptors (LH-R); (II) gonadotrophin-releasing hormone (GnRH) stimulation tests were performed in 10 neutered ferrets, with measurement of androstenedione, 17alpha-hydroxyprogesterone and cortisol as endpoints; (III) GnRH stimulation tests were performed in 15 ferrets of which 8 had hyperadrenocorticism, via puncture of the vena cava under anesthesia; and (IV) urinary corticoid/creatinine (C/C) ratios were measured at 2-week intervals for 1 year in the same ferrets as used in study II. Clear cells in hyperplastic or neoplastic adrenal glands of hyperadrenocorticoid ferrets stained positive with the LH-R antibody. Plasma androstenedione and 17alpha-hydroxyprogesterone concentrations increased after stimulation with GnRH in 7 out of 8 hyperadrenocorticoid ferrets but in only 1 out of 7 healthy ferrets. Hyperadrenocorticoid ferrets had elevated urinary C/C ratios during the breeding season. The observations support the hypothesis that gonadotrophic hormones play a role in the pathogenesis of hyperadrenocorticism in ferrets. This condition may be defined as a disease resulting from the expression of LH-R on sex steroid-producing adrenocortical cells.

Immunohistochemical detection of transforming growth factor-α in Leydig cells during the development of the rat testis

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of transforming growth factor-β1 and -β2 during follicular development in the adult rat ovary

The transforming growth factors-β (TGF-β) affect the metabolic activities of each of the cell types in the ovary. In vitro studies using immature rat ovaries have shown the expression of TGF-β1 and/or TGF-β2 mRNA in thecal/interstitial cells and in granulosa cells (Mulheron and Schomberg, 1990; Mulheron et al., 1991). To obtain information on the localization of TGF-β1 and TGF-β2 in the rat ovary in vivo, we have examined the immunohistochemical staining using antibodies specific for either TGF-β1 orTGF-β2. In the adult ovary the immunostaining for TGF-β1 was intense, whereas the staining for TGF-β2 was faint. The pattern of immunostaining for TGF-β1 and TGF-β2 remained constant in the interstitial cell compartment and was not affected by the stage of the oestrous cycle. Since the interstitium surrounds follicles at all stages of development we conclude that TGF-β is not actively involved in regulating the progression of follicles at discrete stages. At the time of antrum formation in the follicle, intense staining for TGF-β1 was observed in thecal cells. Around the preovulatory stage of development, TGF-β1 and TGF-β2 immunoreactivity was also found in the granulosa cells. In the corpus luteum, intense staining for TGF-β1 was found in some areas, whereas other areas were negative. Weak to moderate staining for TGF-β2 was observed. In conclusion, all major cell types show strong immunostaining for TGF-β1 and less intense staining for TGF-β2, confirming an autocrine/paracrine role for TGF-βs in the regulation of ovarian cell growth and function.

The Histone Deacetylase SIRT1 Controls Male Fertility in Mice Through Regulation of Hypothalamic-Pituitary Gonadotropin Signaling

Abstract Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1−/− mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.

The development of rat Leydig cell progenitors in vitro: how essential is luteinising hormone?

UNLABELLED Luteinising hormone (LH) appears to be important for the establishment of the adult-type Leydig cell population. The role of LH in the initial steps of stem Leydig cell/precursor cell differentiation is less clear. The aim of the present study was to elucidate the role of LH in the differentiation of spindle-shaped mesenchymal-like cells into Leydig cell progenitors. Interstitial cells were isolated from rat testes at three different ages reflecting different phases in development, and cultured in the presence of increasing concentrations of LH (ranging from 0.01 to 10 ng/ml). Cells were isolated from 10-day-old rats when stem Leydig cells/precursor cells are abundant; 13-day-old rats when the first 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-positive Leydig cell progenitors have developed in the strain of rats used in this study; and 18-day-old rats just prior to the wave of progenitor proliferation. Immunohistochemistry revealed that before stem Leydig cells differentiate into progenitor cells, they acquire functional LH receptors and become precursor cells. This was confirmed by an in vivo immunohistochemical double-labelling experiment. Addition of LH to the cultures increased the percentage of LH/3beta-HSD-labelled Leydig cell progenitors, while the percentage of cells solely expressing the LH receptor decreased. Cell proliferation was negligible, suggesting that the increase in 3beta-HSD-positive cells is the result of precursor cell differentiation. When interstitial cells were isolated from 13-day-old rats and to a lesser extent from 10-day-old rats, a small proportion of the precursors could develop into progenitor cells independent of the presence of LH. IN CONCLUSION before Leydig stem cells differentiate into 3beta-HSD-positive progenitor cells, they acquire LH receptors and become precursor cells. LH appears to be essential, even at very low doses for the differentiation of these precursor cells into 3beta-HSD-positive progenitors, although a subpopulation of precursor cells can develop into progenitors independently of LH.

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.

Turnover time of Leydig cells and other interstitial cells in testes of adult rats.

The aim of this study was to investigate the turnover of Leydig cells and other interstitial cells in the adult rat testis. Normal adult rats received injections of [3H]thymidine at 9:00 and 21:00 for 2, 5, or 8 days. The percentage of labeled Leydig cells, which was initially low (0.8% +/- 0.2%), gradually increased during treatment to 1.4% +/- 0.3%. The percentage of labeled peritubular cells was considerably higher and increased from 1.4% +/- 0.1% to 3.5% +/- 0.4% during [3H]thymidine treatment. The remaining interstitial cells were the most actively proliferating cells: the percentage of labeled cells increased from 2.4% +/- 0.2% to 7.5% +/- 0.8% during [3H]thymidine treatment. Leydig cells, peritubular cells, and the remaining interstitial cells in the adult rat testis undergo proliferation. By means of a linear regression analysis and an arcsin transformation, an estimation of the time interval needed to replace various types of interstitial cells was obtained. Taking into account the 95% confidence interval, the turnover time of Leydig cells ranged from 142 to 2823 days. The calculated turnover time for the peritubular cells was 85 to 257 days.

Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes In Vitro

Abstract Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes.