Androgenic Capacity Research Articles

Use of mannitol for cold pretreatment of isolated spikes and as a component of nutrient medium for spring barley anther culture in vitro

Aim. The investigation was aimed to compare the efficiency of mannitol as a component of spike cold pretreatment solution and of medium for spring barley haploid production via anther culture in vitro. Methods. Field-grown plants of two genotypes with a contrast androgenic capacity were used as donors. Cut tillers were pretreated in water at 4⁰C for 5 days, while aseptically isolated spikes were kept in 0.3 M mannitol for 10 days at the same temperature. Isolated anthers were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0%); the media with and without 0.1M mannitol differed in solidifying agents. Results. In highly-responsive line DH00-126, there was an almost two-fold increase in mmgreen plant regeneration frequency (from 48 to 58 plants per 100 anthers) in all experimental variants in comparison to the control. In recalcitrant variety Mebere, the combination of tiller pretreatment in water with anther cultivation on inductive starch-gelling media supplemented with 0.1 M mannitol resulted in the highest green plant yield. Conclusions. Both methodical approaches were proved to be applicable to increase the efficiency of haploid production in spring barley anther culture in vitro.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus)

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Descriptive morphometry and stereology in accessing the testis structure and function of the marsupial Philander frenatus (Olfers, 1818) (Didelphimorphia: Didelphidae).

Philander frenatus is an important marsupial for the maintenance and conservation of the Atlantic Rainforest, however, it has biological characteristics that are still little explored. The study of the reproductive biology is an important key to understand the species reproductive strategies and to improve the development of conservation and management activities. The present study aimed to conduct a histological and morphometric investigation of the testis structure and function of P.frenatus. The average body and testicular weight were 445g and 0.74g, respectively, with a gonadosomatic index of 0.17%. The seminiferous tubules occupying 64.95% of the organ, totalising 9.26m per gram of testis. The tubulesomatic and epitheliumsomatic indexes were 0.10% and 0.07%, respectively. Philander frenatus showed cell loss of approximately 98% during the proliferative phase and the spermatogenic yield was 10.3 cells. The high loss during the mitotic phase contributed to the low spermatogenic yield. The testicular parenchyma was composed of 35% of intertubular components, one of the highest proportions observed in mammals. Leydig cells were responsible for approximately 25% of the testes, followed by lymphatic space (6.44%), blood vessels and connective tissue (4% together). The organisation of the intertubular components resembles the Fawcett III category. The volume and number of Leydig cell per gram of testis were 2,627.12µm3 and 91.28×106 cells, respectively. High investment in the intertubular compartment, specifically number and volume of Leydig cells in P.frenatus is consistent with territorial behaviour and polygynic mating system, which have greater androgenic capacity.

Effect of D-Limonene on the Age-Related Androgenic Changes in Male Rats

Abstract Background: Aging has deteriorating effects on physio-logical functions of many organs including the sex organs, which are suggested to be due to oxidative stress. D-limonene which is an extract from the peel of Citrus plants, has multiple therapeutic properties, mainly due to its antioxidant capacity. Aim of Study: Was to detect the androgenic changes in aging male rats and trying to find out the possible beneficial effects of D-limonene on these androgenic changes. Material and Methods: 32 male rats were divided into 2 main groups; adult sexually mature group and old aged group. Each group was subdivided into; control subgroup and sub-group supplemented with D-limonene for 4 weeks. Blood samples, dissected testes and epididymides were used for detection of oxidative stress markers, sperm analysis, androgen receptors by immunohistochemistry and histopathological examination. Results: The control old aged rats showed significant decrease in all sperm parameters, androgen receptors, antioxi-dants and testosterone levels as compared with the adult ones. Amazingly, supplementation of D-limonene corrected all these aging inducing effects on androgenic capacity which may be attributed to its antioxidant ability. This improving effect was proved by more spread of androgen receptors in rats' epidi-dymides with improving count and morphology of sperms. Conclusion: Supplementation of D-limonene daily to rats, especially the old aged ones could protect the sexual organs from aging oxidative stress inducing deterioration of its physiological functions. So, we recommend in this study to add limonene to the diet of old age men and those who exposed to chronic stress in their lives to save, to some extent, their androgenic capacity which may help in treating many cases of male infertility.

Effects of season, genotype, and nutrient medium on pepper anther culture and microspore developmen

Pure lines of 3 pepper genotypes, including one tolerant (Alata 421) and one intolerant (Alata 195) to lower temperatures and one intolerant (Alata 277A) to higher temperatures, together with one variety (Inan3363) tolerant to higher temperatures, were selected from the pepper collection of Alata Horticultural Research Institute (Mersin, Turkey). Two different culture media (Medium 1 - Murashige and Skoog [MS] + 4 mg L-1 naphthalene acetic acid [NAA] + 0.1 mg L-1 6-benzylaminopurine [BAP] + 0.25% activated charcoal + 8 g L-1 agar + 30 g L-1 sucrose + 15 mg L-1 silver nitrate [AgNO3]; Medium 2 - MS + 4 mg L-1 NAA + 0.5 mg L-1 BAP + 0.25% activated charcoal + 8 g L-1 agar + 30 g L-1 sucrose + 15 mg L-1 AgNO3) were used in this study. The anthers were cultured during different periods (12 months) in order to determine the highest embryo formation and haploid plant regeneration. In addition, all anthers were taken before culturing and on the 1st, 2nd, 3rd, 4th, 8th, and 14th days of different months and were fixed with Carnoy's solution. Development of microspores was observed cytologically using 4'-6-diamidino-2-phenylindole-2HCl (DAPI) and with acetocarmine stain. At the end of the study, it was determined that both embryo formation and haploid plant development varied depending on genotype and cultivation period. No significant differences in the number of embryos obtained were observed between the nutrient media. The highest percentage of haploid embryos was obtained from the Inan3363 variety, known to be tolerant to high temperature. In addition, the androgenic capacity of the Inan3363 variety increased to 65% during some anther culture cultivation periods. The anthers cultured in September and July?August produced the highest yielding results compared with the other periods in terms of number of haploid embryos. Obtaining healthy and developed plants from the embryos was more successful in April, August, September, March, and May than in the other months. Studies on the microspores revealed that the high percentage of uninucleate phase at the beginning of the culture decreased rapidly in the following days.

11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche.

Adrenarche refers to the rise of dehydroepiandrosterone sulfate (DHEA-S) associated with the development of a functional adrenal zona reticularis. Clinical features of adrenarche include onset of body odor, axillary hair, and pubic hair, which reflect increased androgen action. An early rise in adrenal androgens, or premature adrenarche (PremA), is a risk factor for adverse metabolic profiles in adolescence and adulthood. The bioactive androgens associated with adrenarche and PremA remain poorly understood. The adrenal gland is a potential source of testosterone (T) and the 11-oxygenated derivatives 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT). The objective of this study was to characterize the adrenal androgen biome contributing to adrenarche and PremA. With the use of mass spectrometry, 19 steroids including the 11-oxygenated derivatives of T were measured in sera obtained from girls with PremA (n = 37; 4 to 7 years) and age-matched girls (n = 83; 4 to 10 years). In reference population girls, dehydroepiandrosterone, DHEA-S, androstenediol-3-sulfate, T, and 11KT all increased at the onset of adrenarche (6 to 8 years) and beyond (9 to 10 years) (P < 0.05 vs younger subjects 4 to 5 years). T, 11OHT, and 11KT were further elevated in PremA vs age-matched girls (P < 0.001). Circulating concentrations of 11KT during adrenarche and PremA exceeded those of T and 11OHT (11KT > T ≥ 11OHT). Androgen receptor activity and nuclear translocation studies demonstrated that 11KT is a potent androgen similar to T. Our findings suggest that 11KT is the dominant bioactive androgen in children during adrenarche and PremA. Its androgenic capacity suggests that it may be responsible for the phenotypic changes seen in these phenomena.

Genetic Loci Governing Androgenic Capacity in Perennial Ryegrass (Lolium perenne L.).

Immature pollen can be induced to switch developmental pathways from gametogenesis to embryogenesis and subsequently regenerate into homozygous, diploid plants. Such androgenic production of doubled haploids is particularly useful for species where inbreeding is hampered by effective self-incompatibility systems. Therefore, increasing the generally low androgenic capacity of perennial ryegrass (Lolium perenne L.) germplasm would enable the efficient production of homozygous plant material, so that a more effective exploitation of heterosis through hybrid breeding schemes can be realized. Here, we present the results of a genome-wide association study in a heterozygous, multiparental population of perennial ryegrass (n = 391) segregating for androgenic capacity. Genotyping-by-sequencing was used to interrogate gene- dense genomic regions and revealed over 1,100 polymorphic sites. Between one and 10 quantitative trait loci (QTL) were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Most traits were under polygenic control, although a major QTL on linkage group 5 was associated with green plant regeneration. Distinct genetic factors seem to affect green and albino plant recovery. Two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified. Our results shed the first light on the molecular mechanisms behind perennial ryegrass microspore embryogenesis and enable marker-assisted introgression of androgenic capacity into recalcitrant germplasm of this forage crop of global significance.

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (Lolium perenne L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ = 0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.

Effect of genotypical factors on the effectiveness of anther culture in eggplant (Solanum melongena L.)

The aim of this study was to determine the androgenic capacity of some eggplant (Solanum melongena L.) varieties and breeding lines, and to establish the effectiveness of androgenesis induction in the less responsive genotypes by crossing with more responsive genotypes that have a high ability for haploid embryo formation. Flower buds containing greenish-yellow anthers were collected when the microspores were at the late uninucleate stage, which is equivalent to a bud size where petals are not visible. The anthers were kept in the dark at 35 °C for 8 days, then incubated with a 12 h light period at 25 °C for 4 days on C medium, and then transferred to solid R medium, supplemented with 30 g/L sucrose and 0.1 mg/L kinetin. Topan, Halep Karasi, and Teorem F1 varieties and 2 breeding lines [Vd-1 and Vd-2 (LS 2346)] that have a tolerance to Verticillium dahliae Kleb. were used as the plant materials during the first part of the study. Haploid embryos were obtained from Topan and Halep Karasi at the rate of 4.16% and 2.63%, respectively. The responses of Topan and Halep Karasi eggplant varieties to the anther culture were better than those of the Teorem F1 cultivar and the Vd-1 and Vd-2 lines. Because of their responses to anther culture, the Topan and Halep Karasi varieties were used as donor parents, and they were crossed with the other 3 genotypes (Teorem F1, Vd-1, and Vd-2) reciprocally. Gametic embryogenesis among hybrids was only obtained from the Topan × Teorem F1 and Teorem F1 × Topan combinations (0.87% and 2.57%, respectively). Development of the haploid embryos and plant formation occurred at the rates of 0.69% and 2.57%, respectively. This study revealed that the effectiveness of androgenesis in eggplant strongly depended on genotypes. This crossing technique could be useful for increasing the opportunity for haploid embryogenesis from genotypes that are unlikely to form haploid embryos, and crossing between the fair-responsive and poor/nonresponsive genotypes could be a good system for qualifying androgenic response in eggplant.

Enhanced Thecal Androgen Production Is Prenatally Programmed in an Ovine Model of Polycystic Ovary Syndrome

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles (>4 mm) secreted more LH-stimulated androstenedione after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

Study of androgenesis and spontaneous chromosome doubling in barley ( Hordeum vulgare L.) genotypes using isolated microspore culture

This research aimed to study the androgenesis and spontaneous chromosome doubling of five barley genotypes using an isolatedin vitromicrospore culture technique, involving a completely randomized design (CRD) with three replications. Statistical analysis of embryogenesis and cytogenetic results showed that genotype had a significant effect on haploid embryogenesis (P&lt;0.01) and on spontaneous chromosome doubling (P&lt;0.05). The genotype Igri was found to have the highest potential to produce haploid embryos (1577 embryos from 100 anthers), followed by the genotypes Boyer/Rojo, Afzal/Turkman/Kavir, Ashar/Hebo and Agrigashar/Matico with 369, 304, 278 and 150 embryos from 100 anthers, respectively. The highest percentage of spontaneous chromosome doubling (76%) was observed for the genotype which had the lowest embryogenesis (Agrigashar/Matico) and the lowest (65%) for the genotype with the highest androgenic capacity (Igri). Microspore embryogenesis also showed comparatively higher genotypic (109.2) and phenotypic (109.5) coefficients of variation, heritability (99.62) and genetic advance (1206.77), indicating the pre-dominance of additive gene action in the control of this character in the material studied. Estimates of genetic parameters (PCV, GCV and heritability) for microspore embryogenesis were higher than for spontaneous doubled haploids. These results indicated that selection for androgenic capacity would be more effective than for spontaneous doubled haploids. The findings showed a negative relationship (r= −0.68) between embryogenesis and spontaneous chromosome doubling in the barley genotypes studied. All the large embryos used had high regenerability and good plantlet formation.

Steroidogenic Capacity of Residual Ovarian Tissue in 4-Vinylcyclohexene Diepoxide-Treated Mice1

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.

Androgen-dependent morphology of prostates and seminal vesicles in the Hershberger Assay: Evaluation of immunohistochemical and morphometric parameters

The aim of this study was to evaluate androgen-like effects using immunohistochemical and morphometric methods. Therefore, orchiectomized Wistar rats (n > or = 13) were treated s.c. with 1 mg/kg bw/day testosterone propionate (TP) for 7 days and compared to orchiectomized rats without TP substitution (OX) and to an untreated intact control group. Sections obtained from prostates and seminal vesicles were stained with polyclonal and monoclonal antibodies against the androgen receptor (AR) and assessed densitometrically (intensity of the immunoreaction) and morphometrically (epithelial height, luminal area). TP caused an enhancement of staining intensity and an increase in organ weights, epithelial height and luminal area. The use of proliferation markers (PCNA, MIB-5) showed also a highly significant increase of immunoreactive cells in TP-substituted orchiectomized rats compared with the OX group. Based on the present data, the densitometric analysis of AR-immunoreactivity as well as the assessment of proliferation markers, epithelial height and luminal area proved to be sensitive parameters for the evaluation of androgen effects on prostates and seminal vesicles. In further studies these parameters will be used to test several industrial xenooestrogens as well as phytooestrogens on their possible androgenic capacity.

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.

Effect of Cold Pretreatment and Incubation Temperature on Bread Wheat (Triticum aestivum L.) Anther Culture

Two broadly cultivated bread wheat cultivars (“Acheloos” and “Vergina”) and their F1 hybrid were used to study the effect of spike cold pretreatment and the incubation temperature on bread wheat anther culture. For this, half of the spikes carrying anthers at the early uninuclate stage were pretreated at 4°C for 7 days. Subsequently, the anthers with or without cold pretreatment were cultured on W14 induction medium and half of the Petri dishes were incubated at 28°C, while the other half were kept at 32°C. The statistical analysis performed, revealed a strong genotype effect on androgenic capacity. With the exception of green plant production, no significant temperature effect was noticed in the three tested genotypes, whereas genotype X treatment interaction was significant only in regeneration rate and green plant production. With respect to green plant production cv “Acheloos”, although responded well to all treatments, performed better after cold pretreatment. In contrast the F1 hybrid responded better when it was incubated at 32°C without cold pretreatment. It should be mentioned that no DH plants were obtained from cultivar “Vergina” confirming its poor androgenic capacity. Finally, the highest number of DH plants was produced by the F1. This could be attributed to heterosis. It was concluded that cold pretreatment is not always needed and the production of haploid plants is more genotype dependent than the temperature under which the anthers are cultured.

Development of phenolic compounds in maize anthers ( Zea mays) during cold pretreatment prior to androgenesis

Summary Androgenic capacity has been developed for some time in maize ( Zea mays L.) and cold pretreatment of anthers, e.g. for 14 days at 7°C, is considered necessary to induce androgenesis in this species. During this period, development of anther phenolic content and of IAA protection capacity of a phenolic extract were studied in three genotypes that respond differently to anther culture. Phenolic compounds were analyzed by HPLC and found to be mainly represented by six flavonol heterosides. Their levels were highly variable during the cold treatment, both between and within genotypes. These phenolics modified the in vitro degradation of IAA by IAA-oxidase. The extract from the most responsive genotype to androgenesis, DH5×DH7, gave maximal protection of IAA, whereas the ones from Seneca 60 and A188 (low-responsive and unresponsive, respectively) showed an IAA degradation capacity.

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l-1 maltose, 2.5 mg l-1 BA, 0.5 mg l-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.

Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy

Two winter wheat genotypes (‘Dioszegi 200’ and ‘Mv 15’) were compared for their in vitro androgenic capacity. On average, the induction frequency of embryogenic structures was 71.7% in ‘Dioszegi 200’ and only 4.3% in ‘Mv 15’. The haploid induction ability of the two genotypes differed considerably, with ‘Dioszegi 200’ being much higher. The difference in the in vitro inductability of the microspores may result from genetic differences which are manifested in the survival rate of the microspores during the culture period and their adaptability to in vitro conditions. Special DNA fluorochrornes were suitable for studying the different pathways of in vitro androgenesis. Our data indicate that the repeated equal divisions of the microspore nucleus might lead to pollen embryo formation, and subsequent divisions of the vegetative portion of the pollen grain after the first asymmetric microspore mitosis can result in pollen callus formation.

Developmental pattern of the testicular androgen response to gonadotrophin stimulation in vitro and its modification by chronic hypoprolactinaemia.

Developing male rats were treated chronically with bromocriptine (BR, 3 mg/kg b.w. daily) to maintain severe hypoprolactinaemia throughout postnatal development. This treatment induced a precocious increase in Leydig cell numbers per testis and caused substantial, but age-dependent, modifications of the androgenic responsiveness of incubated hemi-testis preparations to stimulation with hCG. Most conspicuous were: (i) a decrease in sensitivity of the testis to hCG at the approach of adult age (due presumably to reduced responsiveness of the Leydig cells), and (ii) a precocious increase in the steroidogenic maximum of the testis at peripubertal age. This probably resulted from the precocious increase in Leydig cell numbers, which was able to mask the negative consequences of reduced androgenic capacity per Leydig cell. The precocious increase in number of Leydig cells induced by hypoprolactinaemia could have resulted from facilitation of the proliferative action of the high prepubertal LH levels on Leydig cell numbers. There were no clear-cut indications for an important effect of BR-induced changes in LH levels, or of a direct effect of BR on the testis.

Dehydroepieandrosterone (DHA), androstenedione (A) and testosterone (T) plasma levels after rapid adrenal suppression and' stimulation in prepubertal and pubertal boys

The supposed role of adrenal secretions in male puberty was investigated by administering dexamethasone (DXM) 1 mg at 9 p.m., then Synacthen (ACTH) 0.25 mg/m2 at 9 a.m., blood beeing collected at 9 a.m. before and after DXM and 1 h. after ACTH. In 11 boys aged 3 to 6 at pubertal stage I (groupe IA) mean DHA levels were (ng/ml) 0.06 before DXM, less than 0.05 after DXM, 0.09 after ACTH. In 21 boys at pubertal stage I aged 7 to 13 (groupe IB) mean DHA levels were respectively 0.76, 0.35, 1.37. In 10 boys at pubertal stage II (groupe II) mean DHA levels were respectively 1.5, 0.82, 2.4. Mean A levels (ng/ml) were respectively 0.11, 0.05, 0.19 in group IA, 0.24, 0.10, 0.28 in group IB, 0.38 0.25, 0.48 in group II. Mean T levels (ng/ml) were respectively 0.05, 0.06, 0.08 in group IA, 0.11, 0.07, 0.14 in group IB, 1.5, 0.70, 0.58 in group II. In the 2 older groups, the variations were highly significant. These data confirm the rise of androgenic capacity of adrenals before puberty and suggest that a large part of circulating T derives both from direct secretion and indirect production, by adrenals.