A synergistic effect of insulin-like growth factor (IGF-I) on equine luteinizing hormone (eLH)-induced testosterone production from cultured Leydig cells of horses

Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1±0.1 year; 1-1.5 year) and eight post-pubertal (2.88±0.35 years; 2-4 years) stallions during routine castrations at the UC Davis Veterinary Hospital. Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated without (control) or with increasing concentrations of purified pituitary-derived eLH and/or recombinant human IGF-I (rhIGF-I) and incubated under 95% air: 5% CO(2) at 32°C for 24h. After 24h, culture media was collected and frozen at -20°C until analyzed for testosterone by a validated radioimmunoassay (RIA). In pubertal stallions, treatment with both increasing concentrations of rhIGF-I and 5ng/ml of eLH failed to demonstrate a significant difference in testosterone production compared with 5ng/ml of eLH only. However, in post-pubertal stallions, a significant increase in the concentration of testosterone in culture media was observed from Leydig cells treated with various concentrations of rhIGF-I and 1 or 5ng/ml of eLH compared with 1 or 5ng/ml of eLH only. In conclusion, IGF-I has a synergistic effect on eLH-induced testosterone production in cultured equine Leydig cells from post-pubertal but not pubertal stallions.     

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH)

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.     

Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis

Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may have potential as a treatment regime to prevent testicular degeneration. The present study was designed to evaluate the anti-apoptotic effect of IGF-I on cultured equine Leydig cells. Testes were collected from 5 post-pubertal stallions (2-4 years old) during routine castrations. A highly purified preparation of equine Leydig cells was obtained from a discontinuous Percoll gradient. Purity of equine Leydig cells was assessed using histochemical 3β-HSD staining. Equine Leydig cells and selected doses of recombinant human IGF-1 (rhIGF-I; Parlow A.F., National Hormone and Peptide Program, Harbor-UCLA Medical Center) were added to wells of 24 or 96 well culture plates in triplicate and cultured for 24 or 48 h under 95% air:5% CO(2) at 34°C. After 24 or 48 h incubation, apoptotic rate was assessed using a Cell Death Detection ELISA kit. Significantly lower apoptotic rates were observed in equine Leydig cells cultured with 5, 10, or 50ng/ml of rhIGF-I compared with control cells cultured without rhIGF-I for 24h. Exposure to 1, 5, 10 or 50 ng/ml of rhIGF-I significantly decreased apoptotic rate in equine Leydig cells cultured for 48 h. After 48 h incubation, cells were labeled with Annexin V and propodium iodine to determine the populations of healthy, apoptotic, and necrotic cells by counting stained cells using a Nikon Eclipse inverted fluorescence microscope. As a percentage of the total cells counted, significantly lower numbers of apoptotic cells were observed in cells treated with 10 (9%) or 50 ng/ml (10%) of rhIGF-I compared with cells cultured without rhIGF-I (control, 22%). In this study, the results from the two assays indicated that rhIGF-I protected equine Leydig cells from undergoing apoptosis during cell culture for 24h or 48 h. In conclusion, IGF-I may be an important paracrine/autocrine factor in protecting equine Leydig cells from undergoing apoptosis.     

Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses

The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450arom) enzyme that converts C19 androgens to C18 estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the breeding and non-breeding seasons. Tissue was fixed and prepared for immunocytochemistry (ICC), carried out with an antiserum against human placental P450arom. In prepubertal colts, there was distinct immunopositive staining of a similar degree within both the Leydig cell and the seminiferous tubule. Horses in the pubertal group had strong Leydig cell immunopositive staining and a slight degree of positive staining within the seminiferous tubules. Postpubertal stallions exhibited definitive immunopositive staining within Leydig cells but not within the seminiferous tubules. Therefore, P450arom is present within the Leydig cell throughout sexual development. In contrast, the presence of P450arom within the seminiferous tubule based upon ICC appeared to be gone by adulthood, suggesting that an age-dependent shift in the locale of this enzyme as the stallion matures.     

Effects of anabolic steroid (19-nortestosterone) on the secretion of testicular hormones in the stallion.

The aim of this study was to clarify the effect of anabolic steroids on the testicular endocrine function of mature stallions. Mature thoroughbred stallions were treated with 800 mg nandrolone decanoate every 3 weeks for 3 months. After the first treatment, plasma concentrations of LH, immunoreactive inhibin and testosterone decreased rapidly to the nadir. These hormones were maintained at significantly lower concentrations compared with concentrations in intact stallions. Histology of the testicular tissue indicated the arrest of advanced spermatogenesis in the seminiferous tubules and a severe depletion of the number of Leydig cells in the interstitial compartment as a result of treatment. Most of the immunopositive cells for the inhibin alpha-subunit and steroidogenesis enzymes in the interstitial compartment decreased below detectable amounts, whereas immunopositive reactions of inhibin alpha-subunit in the seminiferous tubules were clearly observed. In conclusion, the treatment of mature stallions with nandrolone decanoate caused a decrease in the secretion of ir-inhibin and testosterone from the testis, the depletion of the number of Leydig cells and a decrease below detectable amounts of inhibin alpha-subunit and steroidogenesis enzymes. The concentration of ir-inhibin in the peripheral blood may be a useful marker for the examination of testicular activity in stallions being treated with anabolic steroids.     

The effects of age, season and fertility status on plasma and intratesticular insulin-like growth factor I concentration in stallions

The purposes of this study were to establish the basal plasma and testicular insulin-like growth factor-I (IGF-I) values for stallions ranging in age from 6 months to 23 years and to determine if IGF-I could be used as a marker for declining fertility. Blood and testes were obtained from 28 light horse stallions and colts. Of the 28 stallions, 22 were considered fertile and were categorized by age (<2 y, 5 to 10 y, 11 to 15 y, and 16 to 23 y); 12 age-matched stallions were grouped as to fertility status (fertile, subfertile, infertile); and all 28 stallions were grouped as to season of castration (breeding season vs. non-breeding season). In colts less than 2 years of age, IGF-I concentrations in plasma and testicular extracts were higher (P < 0.01) than in the other age groups and were higher in the breeding season than in the non-breeding season (P < 0.01). No significant differences in plasma or testicular extract concentrations of IGF-I were found among fertility groups. The results of this study demonstrate that plasma and testicular IGF-I levels are high in stallions younger than 2 years of age and then decline and plateau in stallions older than 5 years of age, suggesting that IGF-I may be involved in testicular development. The results allude to a possible seasonal effect on IGF-I production. However, it is difficult to separate true seasonality and the effect of age as only those stallions less than 2 years old exhibited variation between seasons. The IGF-I does not appear to have a direct relationship with declined fertility in the stallions tested, suggesting that IGF-I may not be a reliable biomarker for the diagnosis of subfertility and infertility.     

LH and testosterone responses to five doses of a GnRH analogue (buserelin acetate) in 12-month-old Thoroughbred colts

To determine the responsiveness of the pituitary-gonadal axis of peri-pubertal colts to GnRH, buserelin (0.5, 1, 5, 10 and 40 microg) was given to 13 male Thoroughbred yearlings ( n=3-8 colts per dose). Jugular venous blood samples were taken at -10, 0, 10, 20, 30, 40, 60, 120 and 180 min relative to buserelin administration. Increases (P < 0.05) in LH concentrations occurred in colts that received 5, 10, or 40 microg buserelin, but not in those that received 0.5 or 1 microg. Peak LH concentrations and mean area under the curve were higher (P < 0.05) in colts receiving 40 microg buserelin than in those that received 0.5 or 1 microg. Increases ( P< 0.05) in testosterone concentrations occurred in some, but not all, colts that received 1, 5, 10, or 40 microg buserelin. Neither peak concentration nor area under the curve of testosterone differed significantly among doses of buserelin. The percentage of horses that responded to the buserelin increased with increasing dose, with only the highest dose eliciting LH and testosterone responses in all colts. In conclusion, peri-pubertal colts exhibited a dose-response release of LH following buserelin treatment, but individual colts responded in an "all or nothing" manner, such that each either had an LH response or did not. Some colts that exhibited a significant LH response had no subsequent increase in plasma testosterone concentrations; perhaps the pituitary LH response may not have been great enough to stimulate the Leydig cells in these individuals.     

Testis size and onset of spermatogenesis in Cape mountain zebras (Equus zebra zebra)

Testis mass of adult Cape mountain zebra stallions (mean 70.0 g) was appreciably less than that of other zebra species and domestic horses. The histological appearance of the testes of 11-, 24- and 29-month-old colts was typically prepubertal. Spermatogenic activity of a 4-year-old stallion obtained at the end of summer was at a very low level, while a 4.5-year-old stallion obtained 6 weeks after the winter solstice showed a marked increase in spermatogenesis compared with the 4-year-old. Stallions 6.5-19 years of age collected in different seasons all showed active spermatogenesis.     

Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

In-vitro studies of the development of pituitary and testicular functions in diabetes (C57Bl/KsJ-db/db) mutant mice

The hypothesis that male diabetes mutant mice (C57Bl/KsJ-db/db) are suffering from impairment of testicular steroidogenic function and pituitary LH release was tested. A smaller postpubertal increase of testicular weight and a reduction of plasma testosterone and androstenedione levels by 65% at 17 weeks of age were most obvious from the comparison to homozygous lean controls. The ability of constant amounts of Leydig cells, either in crude interstitial cell or in purified Leydig cell suspensions, to respond to maximal doses of hCG or cyclic AMP-was reduced by at least 40% in adult diabetes mice. This defect could be attributed to a 40% decrease of steroid-17 alpha-monooxygenase activity as compared to lean mice. No differences occurred, however, if Leydig cells were submaximally stimulated. GnRH-stimulated pituitary LH release was not significantly changed. The impairment of testicular steroidogenic function in diabetes mutant mice may represent a further aspect of infertility of these animals and of diabetes mellitus.     

Immunolocalization of aromatase in stallion Leydig cells and seminiferous tubules.

High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome p450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (2-5 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidin-peroxidase complex. Immunoreactivity was detected with diaminobenzidine. Immunofluorescence detection, using fluoroisothiocyanate-conjugated goat anti-rabbit antibodies, was also applied. Specific aromatase immunoreactivity was observed intensely in Leydig cells but also for the first time, to a lesser extent, in the cytoplasm surrounding germ cells at the junction with Sertoli cells. Interestingly, the immunoreactivity in Sertoli cells appears to vary with the spermatogenic stages in the basal compartment (with spermatogonia) as well as in the adluminal one (with spermatids). Relative staining intensity in Leydig and Sertoli cells and testicular microsomal aromatase activity increased with age. The present study in stallions indicates that in addition to Leydig cells, Sertoli cells also appear to participate in estrogen synthesis, and this could play a paracrine role in the regulation of spermatogenesis.     

Key factors in the regulation of fetal and postnatal Leydig cell development

The primary function of testicular Leydig cells is the production of androgens to promote sexual differentiation in the fetus, secondary sexual maturation at puberty, and spermatogenesis in the adult. The fetal and postnatal (adult) populations of Leydig cells differ morphologically and have distinct profiles of gene expression. As postnatal Leydig cells differentiate, they transition through three discrete maturational stages characterized by decreasing proliferative rate and increasing testosterone biosynthetic capacity. In this review, we discuss the development of both fetal and postnatal Leydig cells and review the regulation of this process by some of the key hormones and growth factors.     

Leydig cell insufficiency after testicular irradiation for acute lymphoblastic leukemia

Leydig cell function in 21 boys with acute lymphoblastic leukemia who had been treated by bilateral direct testicular irradiation (12 X 2 Gy) at 8.4 +/- 0.7 years, was evaluated 3.8 +/- 0.4 years after irradiation. At the time of irradiation all were prepubertal and at evaluation 12 were prepubertal and 9 pubertal. Leydig cell insufficiency, indicated by a low plasma testosterone response to chorionic gonadotrophin and/or an increase in basal level of plasma luteinizing hormone, was observed in 19/21 patients. The children who were the youngest at testicular irradiation were more vulnerable. Spontaneous virilization occurred in 3 of the older children and resulted from compensated Leydig cell dysfunction.     

Role of testicular interstitial macrophages in regulating testosterone release in hyperprolactinemia

Hyperprolactinemia-induced hypogonadism has been linked to a dysfunction of the hypothalamus-pituitary-testis axis. The direct inhibitory effects of prolactin on the testicular release of testosterone have also been demonstrated, though their mechanisms remain unclear. Incubation of rat testicular interstitial cells (TICs) with prolactin stimulated the release of testosterone. TICs from rats with anterior pituitary-grafting-induced hyperprolactinemia release lower amounts of testosterone than controls. However, Leydig cells isolated from anterior pituitary-grafted rats release a greater amount of testosterone. These paradoxical observations have remained unexplained. This study examined the roles of testicular interstitial macrophages and of their product, tumor necrosis factor-alpha (TNF-alpha), in regulating Leydig cells under condition of hyperprolactinemia. Hyperprolactinemia was induced by grafting two anterior pituitary glands of rats under the renal capsule. Control animals were grafted with rat cortex tissue. The rats were sacrificed 6 weeks later. TICs and macrophages, and Leydig cells were isolated for in vitro incubation and drugs challenge. Testosterone released by testicular interstitial or Leydig cells was measured by radioimmunoassay. TNF-alpha concentration in the medium of TICs or macrophages was measured by enzyme-linked immunosorbent assay (ELISA). A dose-dependent stimulation of TNF-alpha secretion in the medium of TICs or macrophages by the prolactin challenge was observed. Higher amounts of TNF-alpha were released by TICs in the anterior pituitary-grafted rats than in the control group. In contrast, the release of TNF-alpha by testicular interstitial macrophages isolated from the anterior pituitary- and cortex-grafted groups was quantitatively similar. Challenge with human chorionic gonadotropin did not modify the TNF-alpha release by testicular interstitial macrophages in either group. Challenge of Leydig cells with TNF-alpha inhibited their release of testosterone stimulated by human chorionic gonadotropin, but not their basal testosterone release. These different patterns of testosterone release in TICs versus Leydig cells cultures in anterior pituitary-grafted rats may be due to the influence of testicular interstitial macrophages. These observations correlate with in vivo conditions, where prolactin increases the release of TNF-alpha by testicular interstitial macrophages, which, in turn, decreases the human chorionic gonadotropin-stimulated release of testosterone by Leydig cells. In summary, hyperprolactinemia-induced hypogonadism involves a mechanism of prolactin-originated, macrophage-mediated inhibitory regulation of testosterone release by Leydig cells. TNF-alpha, one of the cytokines secreted by macrophages, may play a key role in this mechanism.     

Maintenance of sexual immaturity in male mice and bucks by immunization against N-terminal peptides of the follicle-stimulating hormone receptor

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.     

25-hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.     

Demonstration of LH inhibitor(s) in sheep and human sera

In mature rat Leydig cells, the testosterone output (24 ng/10(6) Leydig cells/4hrs.) is increased 10 fold by LH; the addition of serum from either control or castrated or hypophysectomized rams inhibits (60%) the LH-stimulated testosterone production. Similarly, the incubation of immature rat Leydig cells with sera from hypophysectomized patients leads to a diminution (70 and 30% respectively) of both basal (0.98 ng) and LH stimulated (3.44 ng) testosterone biosynthesis. These data suggest the existence of an LH inhibitor (or inhibitors) in blood from ram and human; in addition, this substance is not only of testicular origin and is not an LH-related molecule.     

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes

Summary The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.     

The Welfare of Young Polish Konik Horses Subjected to Agricultural Workload

Every year a new group of young Polish Konik colts are separated from the forest herds to be trained in the stable breeding system. The aim of this study was to evaluate how the young Polish Konik horses who had been born in a forest reserve adapt to and tolerate draft work. Two groups of 6 horses each were studied: (a) 3- to 4-year-old colts and (b) 7- to 13-year-old stallions. An effort response was estimated by heart rate (HR) registration and biochemical analysis of hematocrit; blood lactic acid (LA) level; and plasma concentration of glucose, triacylglycerols, uric acid, total protein, and cortisol as well as the activity of creatine kinase and lactate dehydrogenase. The mean HR workload response was significantly higher in the group of colts than in the adult stallions: 141 ± 19.3 bpm versus 124 ± 14.4 bpm, respectively. Blood LA level determined after effort was also significantly higher in colts than in stallions: 2.17 ± 0.42 and 1.40 ± 0.16 mmol/l, respectively. The increases in HR and blood LA levels in the colts were higher than in adult stallions, but such increases did not exceed the values characteristic for young working horses. Therefore, the Polish Konik colts evaluated in this study, and new colts who will be separated from the forest herds and brought to the stables in the future, can be subjected to the same work routine that has been used historically because it is not beyond their capabilities.     

Human prepubertal testicular cells in culture: Steroidogenic capacity, paracrine and hormone control

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1–36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1–7-month-old infants (group 1) and 12–36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 μmol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean ± SE: 6.76 ± 1.86, 7.37 ± 1.82, 61.9 ± 1.86, 5.75 ± 1.74 and 8.51 ± 3.23 pmol/10⁶ cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 ± 1.15, 1.50 ± 2.75, 1.44 ± 2.75, 0.78 ± 1.74 and 3.23 ± 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 ± 0.89, 13.5 ± 1.17 and 51.7 ± 3.23), while progesterone secretion remained unchanged (2.82 ± 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins. Therefore, the early postnatal activation of the human testis might be under multiple pituitary hormone control; and, finally, (5) Sertoli cell tumors can secrete paracrine factors that stimulate steroidogenesis.