Loss of the proapoptotic BH3-only protein BCL-2 modifying factor prolongs the fertile life span in female mice.

The duration of the female fertile life span is influenced by the number of oocytes stored in the ovary as primordial follicles. Cell death, both during ovarian development in the embryo and in the postnatal ovary, plays a critical role in determining how many primordial follicles are established and maintained within the ovary. However, the roles of individual apoptotic regulators in mediating cell death within the ovary have not yet been characterized. In this study, gene targeted mice were used to investigate the role of BCL-2-modifying factor (BMF), a proapoptotic protein belonging to the BH3-only subgroup of the BCL-2 family, in determining the number of primordial follicles maintained in the adult ovary and the length of the fertile life span. Stereological analysis of ovaries showed that Bmf(-/-) mice had significantly more primordial follicles than wild-type (WT) control animals at Postnatal Days 100, 200, 300, and 400 but not at Day 20. No differences were observed between WT and Bmf(-/-) mice in the number of ova shed following ovulatory stimulation with exogenous gonadotropins. Bmf(-/-) females were fertile and produced the same number pups/litter as WT females, but Bmf(-/-) females produced litters more frequently and consequently more offspring than WT females over a 6-mo period. Furthermore, the fertile life span of Bmf(-/-) females was significantly extended compared to WT females. Our findings support an important role for BMF in determining the number of primordial follicles maintained in the ovary throughout adult reproductive life and thus indicate that the length of female fertility may be extended by increasing the number of primordial follicles maintained within the ovary through inhibition of BMF.

Electrochemical oxygen reduction behavior of selectively deposited platinum atoms on gold nanoparticles.

Carbon-supported Pt@Au "core-shell" nanoparticles with varying surface concentration of platinum atoms have been synthesized using a novel redox-mediated synthesis approach. The synthesis technique allows for a selective deposition of platinum atoms on the surface of prefabricated gold nanoparticles. Energy dispersive spectroscopic analyses in a scanning electron microscope reveal that the platinum to gold atomic ratios are close to the nominal values, validating the synthesis scheme. X-ray diffraction data indicate an un-alloyed structure. The platinum to gold surface atomic ratio determined from cyclic voltammetry and copper under-potential deposition experiments reveal good agreement with the calculated values at low platinum concentration. However, there is an increase in non-uniformity in the deposition process upon increasing the platinum concentration. Koutecky-Levich analysis of the samples indicates a transition of the total number of electrons transferred (n) in the electrochemical oxygen reduction reaction from two to four electrons upon increasing the surface concentration of platinum atoms. Furthermore, the data indicate that isolated platinum atoms can reduce molecular oxygen but via a two-electron route. Moreover, successful four-electron reduction of molecular oxygen requires clusters of platinum atoms.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion.

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice.

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok(-/-) mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok(-/-) mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok(-/-) mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eµ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.

GHTD-amide: a naturally occurring beta cell-derived peptide with hypoglycemic activity.

In the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues.

Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply.

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924+/-1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987+/-203, with 200-800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254+/-71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332+/-349-3007+/-322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.

Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse.

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.

Methods for quantifying follicular numbers within the mouse ovary.

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 +/- 286 (S.E.M.) and 2227 +/- 101 primordial follicles, and 137 +/- 25 and 265 +/- 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.

Bioorganometallic chemistry. 13. Regioselective reduction of NAD(+) models, 1-benzylnicotinamde triflate and beta-nicotinamide ribose-5'-methyl phosphate, with in situ generated [CpRh(Bpy)H](+): structure-activity relationships, kinetics, and mechanistic aspects in the formation of the 1,4-NADH derivatives.

Cofactor regeneration; i.e., regiospecific conversion of NAD(+) to 1,4-NADH, has been extensively studied and is a crucial component in the eventual use of 1,4-NADH in a variety of bioorganic synthesis processes involving the formation of chiral organic compounds. We have studied the reduction of a model NAD(+) compound, 1-benzylnicotinamide triflate, 1a, using [CpRh(bpy)(H(2)O)](2+), 2 (Cp = eta(5)-C(5)Me(5), bpy = 2,2'-bipyridyl), as the catalyst precursor and sodium formate (HCO(2)Na) as the hydride source in 1:1 H(2)O/THF and have found exclusive 1-benzyl-1,4-dihydronicotinamide regioselectivity, as was observed previously for natural NAD(+) that provided 1,4-NADH (see: Steckhan et al. Organometallics 1991, 10, 1568). Moreover, a variety of 3-substituted derivatives of 1-benzylpyridinium triflate, in addition to the -C(O)NH(2) group (1a), were also studied to ascertain that this 3-functionality (e.g., -C(O)NHCH(3), -C(S)NH(2), -C(O)CH(3), -C(O)OCH(3), and -CN, 1b,d-g) coordinates to a [CpRh(bpy)H](+) complex to direct the concerted, regioselective transfer of the hydride group from the rhodium to the 4-ring position of the NAD(+) model; all coordinating 3-substituents had relative rates in the 0.9-1.3 range with substrate 1a set to 1.0. If in fact the 3-substituent presented a steric effect [-C(O)NH(CH(2)CH(3))(2), 1c] or was a nonbinding group (-CH(3), 1h; -H, 1i), no catalytic hydride transfer was observed even with the more electrophilic 2 and 6 ring positions being readily available, which further implicated the crucial coordination of the NAD(+) model to the CpRh metal ion center. We also found that the 1-benzyl substituent on the nitrogen atom exerted a substantial electron-withdrawing effect, in comparison to the electron-donating 1-methyl substituent, and favorably affected the rate of the regioselective reduction (rate enhancement of 1-benzyl/1-methyl = 2.0). The kinetics of the regioselective reduction of 1a were studied to show that the initial rate of reduction, r(i), is affected by the concentrations of the substrate, 1a, precatalyst, 2, and the hydride source, HCO(2)Na, in 1:1 H(2)O/THF: d[1-benzyl-1,4-dihydronicotnamide]/dt = k(cat)[1a][2][HCO(2)Na]. Furthermore, we wish to demonstrate that a previously synthesized aqueous NAD(+) model, beta-nicotinamide ribose-5'-methyl phosphate, 3, shows a similar regioselectivity for the 1,4-NADH analogue, while the initial rate (r(i)) for the regioselective reduction of 3 and NAD(+) itself was found to be comparable in water but faster by a factor of approximately 3 in comparison to 1a in 1:1 H(2)O/THF; the solvent, THF, appeared to inhibit the rate of reduction in 1a by presumably competing with the substrate 1a for the CpRh metal ion center. However, in H(2)O, the initial kinetic rate for substrate 3 was not affected by its concentration and implies that, in H(2)O, [CpRh(bpy)H](+) formation is rate determining. We assume that binding of 3 and NAD(+) to the CpRh metal ion center is also a pertinent step for 1,4-dihydro product formation, the experimental rate expression in H(2)O being d[1,4-dihydro-beta-nicotinamide ribose-5'-methyl phosphate]/dt = k(cat)[2][HCO(2)Na]. What we have discovered, for the first time, is evidence that the regioselective reduction of NAD(+) to 1,4-NADH by [CpRh(bpy)H](+) is a consequence of the amide's ability to coordinate to the CpRh metal center, thereby constricting the kinetically favorable six-membered ring transition state for plausible concerted hydride transfer/insertion to C4 to regioselectively provide the 1,4-NADH derivative; [CpRh(bpy)H](+) can be categorized as a biomimetic enzymatic hydride via its ability to bind and regioselectively transfer hydride to C4, exclusively. Clearly, the pyrophosphate and adenosine groups associated with the structure of NAD(+) are not essential in the rate of hydride transfer to C4, with NAD(+) model 3 having a similar initial rate (r(i)) of reduction as NAD(+) itself in water. Finally, a catalytic cycle will be proposed to account for our overall observations.

The road to ovulation: the role of oestrogens.

Oestrogens have been known for many years to have a direct influence on folliculogenesis. Oestradiol-17beta (E2) and its analogues have both proliferative and differentiative effects on somatic cells of follicles. Nevertheless, definitive proof of an obligatory role for oestrogen in folliculogenesis and elucidation of the mechanisms subserving its different actions in follicular cells remains elusive. Several recent developments permit a re-examination of the roles and actions of E2 in the follicle. They are: (i) the discovery of a second form of the oestrogen receptor, ERbeta; (ii) the advent of genetically modified mice with deletions in the ERalpha (alphaERKO) ERbeta (BERKO) and the double ER deletions (alphabetaERKO); and (iii) a mouse model of oestrogen deficiency (ArKO) by targeted disruption of the cyp 19 gene encoding the aromatase enzyme. Recent information derived from these models is reviewed to re-assess the roles and actions of oestrogens in follicular dynamics and the phenotypic differentiation of ovarian somatic cells in the ovary. The data demonstrate that oestrogen is obligatory for normal folliculogenesis and that the phenotype of the ovarian somatic cells depends on the steroid milieu. The ArKO mouse provides a model to test the roles of the respective ERs in proliferation and differentiation using specific agonists and antagonists, and to study regulation of the differentiation of ovarian and testicular somatic cells.

Estrogenic induction of spermatogenesis in the hypogonadal mouse.

Abnormal sperm production and reduced fertility have been reported in transgenic male mice lacking the alpha-subtype of the estrogen receptor (ER)alpha or aromatase. The aim of this study was to investigate the role of estrogen in male reproductive function, by determining the effect of estradiol on testicular function in hypogonadal (hpg) mice congenitally lacking gonadotropin; and thus, sex steroid production. hpg mice were treated, at 2-3 months of age, with slow-release estradiol implants, which achieved circulating estradiol concentrations of approximately 40 pg/ml. Treatment for 35 days reliably induced a 4- to 6-fold increase in testicular weight, compared with the vestigial testes in the untreated or cholesterol-treated controls. The degree of testicular growth after 35 days was similar to that in hpg mice receiving an intrahypothalamic graft of preoptic area tissue taken from neonatal mice on the day of birth, a procedure known to induce testicular development in hpg mice by activation of the pituitary gland. Histological analysis revealed that the testes contained elongated spermatids after 35 days of estradiol treatment, whereas germ cell development never progressed beyond the pachytene stage in control hpg mice. Treatment for 70 days induced full qualitatively normal spermatogenesis in hpg mice. Testis weight increased 5-fold, reflecting a 5-fold increase in total seminiferous tubule volume and a 4- to 5-fold increase in the total volume of the seminiferous epithelium. In all experiments, spermatogenesis proceeded in the absence of measurable androgen concentrations, but circulating FSH concentrations were slightly (but significantly) elevated, relative to cholesterol-treated control hpg mice. This stimulatory action of estradiol on FSH secretion was unexpected, particularly because identical estradiol treatments significantly decreased serum FSH levels in wild-type littermates. These results indicate that estrogens may play a role in spermatogenesis, via stimulatory effects on FSH secretion. An alternative or complementary explanation, given the recent identification of estrogen receptors (ERalpha and ERbeta) and aromatase within various cell types in the testis, is that estrogens exert paracrine actions within the testis to promote spermatogenesis. The identification of effects of estradiol on testicular function provides a conceptual basis to reexamine the speculative link between increased exposure to environmental estrogens and reduced fertility in man.

Luteal regression in the primate: different forms of cell death during naturaland gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis.

Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.

Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development.

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.

Investigation of the wavelength accuracy of brewer spectrophotometers.

The wavelength accuracy of the Brewer spectrophotometer in the 280-360-nm spectral range is improved by a new grating-drive mechanism and a new dispersion function derived from the Brewer geometry. With the new mechanism, the reproducibility of wavelength settings for spectral emission lines is better than 0.3 pm (0.0003 nm) and, with a thermally compensated version, the effect of temperature is less than 0.4 pm K(-1). The new dispersion function fits spectral line positions better than the standard function and is less prone to extrapolation error. Applying the new function to data from four new and ten standard drives shows that the new drives perform as well as the best of the standard ones and much better than the majority.

Macro, micro, and molecular research on spermatogenesis: the quest to understand its control.

Synchronous maturation of the germ cells in the seminiferous epithelium has long been recognized by microscopy, and is believed to be a consequence of a complex interaction between the germ cells and the Sertoli cells, largely driven by testosterone and its synergistic action with follicle-stimulating hormone. Overall coordination of the cycle of the seminiferous epithelium is reviewed with regard to the known and possible actions of testosterone upon the Sertoli cells and the germ cells. With gradual refinements of optical instrumentation and development of a wide range of histological, morphometric, biochemical, and molecular techniques, coupled with selective alterations of hormonal stimulation and the cellular composition of the testis, new approaches to the question of how sperm production is regulated are becoming available. Germ cell and Sertoli cell functions are intimately related to each other via local, intratesticular or paracrine signals which are suppressed or triggered at certain defined steps in the spermatogenic process. The coordination of germ cell proliferation and maturation is discussed in terms of the contributions made by microscopical techniques.

Effect of seminiferous tubule size on hCG-induced regeneration of peritubular Leydig cells in hypophysectomized, EDS-treated rats.

Following their selective destruction 3 weeks previously by administration of ethane dimethanesulphonate (EDS) the regenerative capacity of Leydig cells was assessed in relation to seminiferous tubule morphology in hypophysectomized adult rats administered 7 daily injections of 100 iu hCG. Total Leydig cell volume per testis in hCG-treated rats (30.2 +/- 3.2 microliters, mean +/- SEM) was significantly (p < 0.01) greater than in the testes of rats at 3 and 4 weeks after EDS-treatment (7.6 +/- 0.7 and 22.7 +/- 1.4 microliters, respectively). Regeneration of Leydig cells in hCG-treated rats significantly (p < 0.05) favoured peritubular locations (18.6 +/- 2.8 microliters/testis) compared to central or perivascular sites of origin (11.6 +/- 1.2 microliters/testis). Partial restoration of spermatogenesis occurred in hCG-treated rats (tubule diameters usually > 250 microns) and a significant inverse correlation was found between peritubular Leydig cell percentage, or total volume per testis, and the volumetric proportion of seminiferous tubules (r = -0.94, p < 0.001) or the seminiferous epithelium (r = -0.73 to -0.79, p < 0.05-0.01). No significant (p > 0.4-0.9) correlation existed between centrally-regenerated Leydig cells and these parameters. The results show that in response to hCG stimulation, Leydig cells are more likely to develop around smaller seminiferous tubules, suggesting that hCG alone cannot mimic the expected pattern of Leydig cell regeneration (central and peritubular origins) which occurs during normal sexual maturation or at 3-4 weeks after EDS treatment. It is concluded that other factors, possibly FSH, are required for typical Leydig cell development which in turn may be influenced by local cellular growth factors originating from either the seminiferous tubules or the adjacent intertubular tissue.

Temporal relationship between androgen-dependent changes in the volume of seminiferous tubule fluid, lumen size and seminiferous tubule protein secretion in rats.

This study assessed whether changes in production of seminiferous tubule fluid underlie the previously described androgen-dependent changes in protein secretion by seminiferous tubules at stages VI-VIII of the spermatogenic cycle. Testosterone withdrawal was induced in adult rats by administration of ethane dimethane sulfonate (EDS) and temporal changes in lumen area, the volume of seminiferous tubule fluid and testicular interstitial fluid were assessed and compared with the changes in secretion of [35S]methionine-labelled proteins in vitro by isolated seminiferous tubules at stages VI-VIII. Testicular interstitial fluid was reduced by about 50% by day 4 and later after EDS treatment when compared with controls. In contrast, the volume of seminiferous tubule fluid was unaffected at days 3 and 4 but was reduced by about 50% at days 6 and 8 after EDS treatment. In perfusion-fixed control testes, the lumen area of seminiferous tubules at stages VII-VIII was significantly greater than at stages I-VI and IX-XIV. This difference was also evident at 4 days after EDS treatment, but was abolished at 6 and 8 days after treatment. The volume of testicular interstitial fluid was reduced significantly at 3 and 4 days after EDS treatment, but was further reduced (about 50%) at 6 and 8 days. Administration of 25 mg testosterone esters every 3 days to EDS-treated rats prevented all of the changes described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion.

Spectral measurements of ultraviolet-B radiation made at Toronto since 1989 indicate that the intensity of light at wavelengths near 300 nanometers has increased by 35 percent per year in winter and 7 percent per year in summer. The wavelength dependence of these trends indicates that the increase is caused by the downward trend in total ozone that was measured at Toronto during the same period. The trend at wavelengths between 320 and 325 nanometers is essentially zero.