Physiological and metabolic aspects of follicular developmental competence as affected by lactational body condition loss.

Metabolic demands of modern hybrid sows have increased over the years, which increases the chance that sows enter a substantial negative energy balance (NEB) during lactation. This NEB can negatively impact reproductive outcome, which is especially evident in primiparous sows causing a reduced second parity reproductive performance. The negative effects of the lactational NEB on reproductive performance can be partly explained by the influence of the premating metabolic state, during and after lactation, on the development of follicles from which oocytes will give rise to the next litter. In addition, the degree and type of body tissue mobilization during lactation that is, adipose tissue or lean mass, highly influences follicular development. Research investigating relations between the premating metabolic state and follicular and oocyte competence in modern hybrid sows, which experience higher metabolic demands during lactation, is limited. In this review we summarize current knowledge of physiological relations between the metabolic state of modern hybrid sows and follicular developmental competence. In addition, we discuss potential implications of these relations for current sow management strategies.

Effects of Bisphenol A on reproductive toxicity and gut microbiota dysbiosis in male rats.

Bisphenol A (BPA) is an environmental endocrine disruptor. Recent studies have shown an association between decreased spermatogenesis and gut microbiota alteration. However, the potential associations and mechanisms of BPA exposure on spermatogenesis, hormone production, and gut microbiota remain unknown. This study aims to investigate BPA-induced male reproductive toxicity and the potential link with gut microbiota dysbiosis. Male Sprague Dawley rats were exposed to BPA at different doses by oral gavage for thirty consecutive days. The extent of testicular damage was evaluated by basic parameters of body weight and hematoxylin-eosin (H&E) staining. Next, we determined the mRNA levels and protein levels of apoptosis, histone-related factors, and mammalian target of rapamycin (mTOR) pathway in testes. Finally, 16 S rDNA sequencing was used to analyze gut microbiota composition after BPA exposure. BPA exposure damaged testicular histology, significantly decreased sperm count, and increased sperm abnormalities. In addition, BPA exposure caused oxidative stress and cell apoptosis in testes. The levels of histone (H2A, H3) were significantly increased, while ubiquitin histone H2A (ub-H2A) and ubiquitin histone H2B (ub-H2B) were markedly reduced. Furthermore, BPA activated the PI3K and AKT expression, but the protein expressions of mTOR and 4EBP1 in testes were inhibited significantly. Additionally, the relative abundance of class Gammaproteobacteria, and order Betaproteobacteriales was significantly higher when treated with a high dose of BPA compared to the control group, which was negatively correlated with testosterone level. This study highlights the relationship between BPA-induced reproductive toxicity and gut microbiota disorder and provides new insights into the prevention and treatment of BPA-induced reproductive damage.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = -6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

Pseudo-Starvation Driven Energy Expenditure Negatively Affects Ovarian Follicle Development.

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis.

There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration.Atretic preantal and antral follicles were investigated using immunohistochemistry and laser-capture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy.

Dietary-Induced Chronic Hypothyroidism Negatively Affects Rat Follicular Development and Ovulation Rate and Is Associated with Oxidative Stress.

The long-term effects of chronic hypothyroidism on ovarian follicular development in adulthood are not well known. Using a rat model of chronic diet-induced hypothyroidism initiated in the fetal period, we investigated the effects of prolonged reduced plasma thyroid hormone concentrations on the ovarian follicular reserve and ovulation rate in prepubertal (12-day-old) and adult (64-day-old and 120-day-old) rats. Besides, antioxidant gene expression, mitochondrial density and the occurrence of oxidative stress were analyzed. Our results show that continuous hypothyroidism results in lower preantral and antral follicle numbers in adulthood, accompanied by a higher percentage of atretic follicles, when compared to euthyroid age-matched controls. Not surprisingly, ovulation rate was lower in the hypothyroid rats. At the age of 120 days, the mRNA and protein content of superoxide dismutase 1 (SOD1) were significantly increased while catalase (CAT) mRNA and protein content was significantly decreased, suggesting a disturbed antioxidant defense capacity of ovarian cells in the hypothyroid animals. This was supported by a significant reduction in the expression of peroxiredoxin 3 ( ITALIC! Prdx3), thioredoxin reductase 1 ( ITALIC! Txnrd1), and uncoupling protein 2 ( ITALIC! Ucp2) and a downward trend in glutathione peroxidase 3 ( ITALIC! Gpx3) and glutathione S-transferase mu 2 ( ITALIC! Gstm2) expression. These changes in gene expression were likely responsible for the increased immunostaining of the oxidative stress marker 4-hydroxynonenal. Together these results suggest that chronic hypothyroidism initiated in the fetal/neonatal period results in a decreased ovulation rate associated with a disturbance of the antioxidant defense system in the ovary.

Development and application of a health-based framework for informing regulatory action in relation to exposure of microplastic particles in California drinking water.

Microplastics have been documented in drinking water, but their effects on human health from ingestion, or the concentrations at which those effects begin to manifest, are not established. Here, we report on the outcome of a virtual expert workshop conducted between October 2020 and October 2021 in which a comprehensive review of mammalian hazard studies was conducted. A key objective of this assessment was to evaluate the feasibility and confidence in deriving a human health-based threshold value to inform development of the State of California's monitoring and management strategy for microplastics in drinking water. A tiered approach was adopted to evaluate the quality and reliability of studies identified from a review of the peer-reviewed scientific literature. A total of 41 in vitro and 31 in vivo studies using mammals were identified and subjected to a Tier 1 screening and prioritization exercise, which was based on an evaluation of how each of the studies addressed various quality criteria. Prioritized studies were identified largely based on their application and reporting of dose-response relationships. Given that methods for extrapolating between in vitro and in vivo systems are currently lacking, only oral exposure in vivo studies were identified as fit-for-purpose within the context of this workshop. Twelve mammalian toxicity studies were prioritized and subjected to a Tier 2 qualitative evaluation by external experts. Of the 12 studies, 7 report adverse effects on male and female reproductive systems, while 5 reported effects on various other physiological endpoints. It is notable that the majority of studies (83%) subjected to Tier 2 evaluation report results from exposure to a single polymer type (polystyrene spheres), representing a size range of 0.040 to 20 µm. No single study met all desired quality criteria, but collectively toxicological effects with respect to biomarkers of inflammation and oxidative stress represented a consistent trend. While it was possible to derive a conservative screening level to inform monitoring activities, it was not possible to extrapolate a human-health-based threshold value for microplastics, which is largely due to concerns regarding the relative quality and reliability of current data, but also due to the inability to extrapolate data from studies using monodisperse plastic particles, such as polystyrene spheres to an environmentally relevant exposure of microplastics. Nevertheless, a conservative screening level value was used to estimate a volume of drinking water (1000 L) that could be used to support monitoring activities and improve our overall understanding of exposure in California's drinking water. In order to increase confidence in our ability to derive a human-health-based threshold value in the future, several research recommendations are provided, with an emphasis towards strengthening how toxicity studies should be conducted in the future and an improved understanding of human exposure to microplastics, insights critically important to better inform future risk assessments. Supplementary Information: The online version contains supplementary material available at 10.1186/s43591-022-00030-6.

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene.

Beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 (+/+)) mice efficiently cleave BC. Bcmo1 (-/-) mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 (-/-) mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 (-/-) mice and Bcmo1 (+/+) mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 (-/-) mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 (-/-) mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 (-/-) mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.

Chronic hypothyroidism only marginally affects adult-type Leydig cell regeneration after EDS administration.

Chronic prenatally induced dietary hypothyroidism delays adult-type Leydig cell development, but does not block this process. Using a chemical model to induce hypothyroidism, it was suggested that development of a new population of Leydig cells was completely inhibited following the addition of the cytotoxic compound ethane-1,2-dimethyl sulphonate (EDS). In this study, we used a dietary approach to induce hypothyroidism and reinvestigated the regeneration of the Leydig cell population following EDS administration. Eighty-four day old euthyroid and chronically hypothyroid rats received an injection of EDS and were killed directly before or at regular intervals up to 77 days after EDS. In some control and hypothyroid animals, the first progenitor-type Leydig cells were observed at day 12 after EDS. At day 16, Leydig cell progenitors were present in all rats. The percentage of proliferating Leydig cells peaked in the euthyroid animals at day 21 after EDS. In the hypothyroid testis such a peak was not observed, although the percentage of proliferating regenerating Leydig cells was significantly higher from days 35 to 56 compared with the controls. This suggested that the wave of Leydig cell proliferation was delayed in the hypothyroid animals as compared with the euthyroid controls. On the day of EDS injection, the Leydig/Sertoli cell ratio was 37% lower in the hypothyroid rats compared with the controls. The Leydig/Sertoli cell ratio remained lower in the EDS-treated hypothyroid animals compared with the controls at all time points investigated. At day 77 after EDS, the Leydig cell population had returned to its pre-treatment size in both groups. Plasma testosterone production was reduced to below detectable levels immediately after EDS injection, and started to increase again on day 16, reaching pre-treatment values on day 21 in both groups. Taken together, severely reduced thyroid hormone levels did not block the regeneration of the adult-type Leydig cell population following EDS, as has been suggested previously.

Transcriptome Analysis of Porcine Granulosa Cells in Healthy and Atretic Follicles: Role of Steroidogenesis and Oxidative Stress.

One of the main causes of female infertility is a deregulated antral follicular atresia, a process of which the underlying molecular mechanisms are largely unknown. Our objective was therefore to characterize the complex transcriptome changes in porcine granulosa cells of healthy antral (HA) and advanced antral atretic (AA) follicles, using ELISA and RNA-Seq followed by qRT-PCR and immunohistochemistry. Granulosa cell RNA-Seq data revealed 2160 differentially expressed genes, 1483 with higher and 677 with lower mRNA concentrations in AA follicles. Bioinformatic analysis showed that the upregulated genes in AA follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes (e.g., GSTA1, GCLC, GCLM, IDH1, GPX8) involved in the glutathione metabolism pathway and other redox-related genes (e.g., RRM2B, NDUFS4). These observations were confirmed by RT-qPCR and immunohistochemistry. Additionally, the granulosa cells of AA follicles express significantly stronger 8-OHdG immunostaining, a marker of oxidative DNA damage, implicating that oxidative stress may participate in follicular atresia. We hypothesize that the decrease in anti-apoptotic factors and steroid hormones coincides with increased oxidative stress markers and the expression of pro-apoptotic factors, all contributing to antral follicular atresia.

A comparative analysis of human adult testicular cells expressing stem Leydig cell markers in the interstitium, vasculature, and peritubular layer.

BACKGROUND: Origin of human adult Leydig cells (ALCs) is not well understood. This might be partly due to limited data available on the identification and location of human precursor and stem Leydig cells (SLCs) which hampers the study on the development of ALCs. OBJECTIVES: The aim of the present study was to investigate whether described human (PDGFRα, NGFR) and rodent (NES, PDGFRα, THY1, NR2F2) SLC markers are expressed by a common cell population within human adult testicular interstitial cells in vivo and before and after in vitro propagation. MATERIALS AND METHODS: Immunohistochemical analyses were used to identify localization of human adult testicular interstitial cells expressing described SLC markers. Next, interstitial cells were isolated and cultured. The percentage of cells expressing one or more SLC markers was determined before and after culture using flow cytometry. RESULTS: NR2F2 and PDGFRα were present in peritubular, perivascular, and Leydig cells, while THY1 was expressed in peritubular and perivascular cells. Although NES and NGFR were expressed in endothelial cells, co-localization with PDGFRα was found for both in vitro, although for NGFR only after culture. All marker positive cells were able to undergo propagation in vitro. DISCUSSION: The partly overlap in localization and overlap in expression in human testicular cells indicate that PDGFRα, NR2F2, and THY1 are expressed within the same ALC developmental lineage from SLCs. Based on the in vitro results, this is also true for NES and after in vitro propagation for NGFR. CONCLUSION: Our results that earlier described SLC markers are expressed in overlapping human interstitial cell population opens up further research strategies aiming for a better insight in the Leydig cell lineage and will be helpful for development of strategies to cure ALC dysfunction.

Steroid profile of porcine follicular fluid and blood serum: Relation with follicular development.

The aim of this study was to identify follicular fluid (FF) steroids which reflect follicular development in the early stages of the follicular phase and to establish whether the levels of these FF steroids correspond to their levels in serum. If these relations are established, serum steroid profiles may be used to monitor follicular development already in this early stage of the follicular phase. We used samples of two experiments, one with multiparous sows at the onset of the follicular phase (weaning) and one with primiparous sows at the midfollicular phase (48 hr after weaning). Complete steroid profiles were measured in pooled FF of the 15 largest follicles and serum using high-performance liquid chromatography-tandem mass spectrometry. In experiment 1, pooled FF volume, as a measure for average follicle size, tended to be positively related to higher FF 17ß-estradiol levels (ß = 0.56, p = .08). In experiment 2, a larger FF volume was related not only to FF higher 17ß-estradiol levels (ß = 2.11, p < .001) but also to higher levels of ß-nortestosterone (ß = 1.15, p < .0001) and its metabolite 19-norandrostenedione (ß = 1.27, p < .01). In addition, FF volume was related to higher FF 17α-OH-pregnenolone (ß = 1.63, p = .03) and 17α-OH-progesterone (ß = 1.83, p < .001), which could indicate that CYP17,20-lyase activity is limiting for 17ß-estradiol production in larger follicles at the beginning of the follicular phase. In serum, most of the steroids were present at lower levels compared to FF, except for the corticosteroids. Serum progestins and androgens were never related to follicle pool volume and steroid levels did not differ in the midfollicular phase compared to the onset of the follicular phase in the second experiment. Serum steroid levels therefore poorly reflect the developmental stage of the follicle pool in the first half of the follicular phase of the estrous cycle in sows.

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.

Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary.

BACKGROUND: Anti-Müllerian hormone (AMH) is expressed by granulosa cells of developing follicles and plays an inhibiting role in the cyclic process of follicular recruitment by determining follicle-stimulating hormone threshold levels. Knowledge of AMH expression in the porcine ovary is important to understand the reproductive efficiency in female pigs. RESEARCH AIM: In the present study we investigated the expression of AMH during follicular development in prepubertal and adult female pigs by immunohistochemistry, laser capture micro-dissection and RT-qPCR. RESULTS AND CONCLUSION: Although in many aspects the immunohistochemical localization of AMH in the porcine ovary does not differ from other species, there are also some striking differences. As in most species, AMH appears for the first time during porcine follicular development in the fusiform granulosa cells of recruited primordial follicles and continues to be present in granulosa cells up to the antral stage. By the time follicles reach the pre-ovulatory stage, AMH staining intensity increases significantly, and both protein and gene expression is not restricted to granulosa cells; theca cells now also express AMH. AMH continues to be expressed after ovulation in the luteal cells of the corpus luteum, a phenomenon unique to the porcine ovary. The physiological function of AMH in the corpus luteum is at present not clear. One can speculate that it may contribute to the regulation of the cyclic recruitment of small antral follicles. By avoiding premature exhaustion of the ovarian follicular reserve, AMH may contribute to optimization of reproductive performance in female pigs.

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.

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro.

BACKGROUND: The initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential. Oncostatin M (OSM) is a multifunctional cytokine and member of the interleukin (IL)-6 family that also includes other cytokines such as LIF. In the rat OSM is highly expressed in the late fetal and neonatal testis, and may thus be a candidate factor involved in Leydig cell progenitor formation. METHODS: Interstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days in the presence of different doses of OSM (range: 0.01 to 10 ng/ml) alone or in combination with LH (1 ng/ml). The effects of OSM and LH on cell proliferation were determined by incubating the cultures with [3H]thymidine or bromodeoxyuridine (BrdU). Developing progenitor cells were identified histochemically by the presence of the marker enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD). RESULTS: OSM, when added at a dose of 10 ng/ml, caused a nearly 2-fold increase in the percentage of Leydig cell progenitors after 8 days of culture. Immunohistochemical double labelling experiments with 3beta-HSD and BrdU antibodies showed that this increase was the result of differentiation of stem Leydig cells/precursor cells and not caused by proliferation of progenitor cells themselves. The addition of LH to the cultures consistently resulted in an increase in progenitor formation throughout the culture period. Surprisingly, when OSM and LH were added together, the LH induced rise in progenitor cells was significantly inhibited after 3 and 8 days of culture. CONCLUSION: Taken together, the results of the present study suggest that locally produced OSM may not only play a role in the regulation of Sertoli cell proliferation and the initiation of spermatogenesis but may also play a role in the regulation of Leydig cell progenitor formation by keeping the augmenting effects of LH on this process in abeyance.

Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development.

Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

Prolonged hypothyroidism severely reduces ovarian follicular reserve in adult rats.

BACKGROUND: There is substantial evidence both in humans and in animals that a prolonged reduction in plasma thyroid hormone concentration leads to reproductive problems, including disturbed folliculogenesis, impaired ovulation and fertilization rates, miscarriage and pregnancy complications. The objective of the present study is to examine the consequences of chronic hypothyroidism, induced in adulthood, for the size of the ovarian follicle pool. In order to investigate this, adult female rats were provided either a control or an iodide deficient diet in combination with perchlorate supplementation to inhibit iodide uptake by the thyroid. Sixteen weeks later animals were sacrificed. Blood was collected for hormone analyses and ovaries were evaluated histologically. RESULTS: At the time of sacrifice, plasma thyroid-stimulating hormone concentrations were 20- to 40-fold increased, thyroxine concentrations were negligible while tri-iothyronin concentrations were decreased by 40% in the hypothyroid group, confirming that the animals were hypothyroid. Primordial, primary and preantral follicle numbers were significantly lower in the hypothyroid ovaries compared to the euthyroid controls, while a downward trend in antral follicle and corpora lutea numbers was observed. Surprisingly the percentage of atretic follicles was not significantly different between the two groups, suggesting that the reduced preantral and antral follicle numbers were presumably not the consequence of increased degeneration of these follicle types in the hypothyroid group. Plasma anti-Müllerian hormone (AMH) levels showed a significant correlation with the growing follicle population represented by the total ovarian number of primary, preantral and antral follicles, suggesting that also under hypothyroid conditions AMH can serve as a surrogate marker to assess the growing ovarian follicle population. CONCLUSIONS: The induction of a chronic hypothyroid condition in adult female rats negatively affects the ovarian follicular reserve and the size of the growing follicle population, which may impact fertility.

Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary.

In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts, though the amount of protein varied with the phase of the estrous cycle. Fas, Bax and caspase-3 protein levels were highest at diestrus and decreased thereafter towards metestrus. In contrast, Fas ligand and Bcl-2 protein levels were lowest at diestrus and increased toward metestrus. Immunohistochemistry revealed that the staining of the anti-apoptotic protein Bcl-2 was more pronounced in healthy preantral follicles than in atretic follicles. In contrast, the pro-apoptotic proteins Fas, Fas ligand, Bax and active caspase-3 were more predominantly present in atretic follicles. In the ovarian surface epithelium (OSE), Fas, procaspase-3 and Bcl-2 immunostaining appeared independent of the phase of the estrous cycle. Fas ligand and Bax staining was detected particularly during proestrus in OSE cells surrounding the ovulatory follicles, while active caspase-3 was observed only in OSE cells at the postovulatory site during estrus. The proportion of luteal cells that stained positively for Fas, Bax and caspase-3 increased with the age of the corpus luteum, while Fas ligand and Bcl-2 immunostaining was strongest in newly formed corpora lutea and decreased thereafter. In conclusion, the components of the Fas signalling pathway were differentially expressed throughout the estrous cycle in a variety of ovarian cell types, which may correspond to hormone dependent survival mechanisms.