Estela Muñoz

SEASONAL CHANGES OF THE LEYDIG CELLS OF VISCACHA (LAGOSTOMUS MAXIMUS MAXIMUS). A LIGHT AND ELECTRON MICROSCOPY STUDY

The Leydig cells of viscacha (seasonal rodent) show cytoplasmic hypertrophy and regional distribution during the breeding period (summer-autumn). The dominant organelles are smooth endoplasmic reticulum (SER) and mitochondria. A moderately well-developed Golgi, abundant lipid inclusions, dense bodies like lysosomes in different stages, and centrioles are observed. Extensive or focal desmosome and gap-like junctions between neighbouring Leydig cells are present. These cells exhibit an evident hypotrophy and an increase in the number of dense bodies during the gonadal regression in winter (July and August). Cells in different stages of involution are observed in this period. Their nuclei are irregular and heterochromatic. The cytoplasm contains few mitochondria. The vesicular SER is scarse. Irregular and large intercellular spaces with microvilli and amorphous material are present. The junctional complexes are absent. The nuclear and cytoplasmic volume and development of SER and mitochondria increase during the recovery period (spring). The lipid inclusions decrease. Dilatations of the intercellular space with microvilli and limited by focal desmosome-like junctions are observed. In conclusion, the Leydig cells of Lagostomus maximus maximus show deep changes alongside the reproductive cycle. The photoperiod variations, through pineal hypothalamus pituitary axis and the hormone melatonin, are probably responsible for them. Moreover, the fall of serum and tubular testosterone would be one of the factors responsible for gonadal regression.

Seasonal Variations in the Expression of the mRNA Encoding ß1-Adrenoceptor and AA-NAT Enzyme, and in the AA-NAT Activity in the Pineal Gland of Vizcacha (Lagostomus maximus maximus) – Correlation With Serum Melatonin

The vizcacha is a photoperiodic rodent living in the southern hemisphere. The adult males exhibit an annual reproductive cycle characterized by a gonadal regression period during winter with, in some animals, an almost complete loss of spermatogenesis. In this study, we investigated whether biochemical parameters involved in melatonin synthesis in the vizcacha pineal gland exhibited an annual rhythm in parallel with the annual reproductive cycle. By use of in situ hybridization, an annual variation of mRNA encoding ß 1 -adrenoceptor was shown, with a maximum during autumn and winter. In situ hybridization for mRNA encoding AA-NAT enzyme also exhibited an annual rhythm with the lowest and highest levels in May and August, respectively. Likewise, in August the activity of arylalkylamine N-acetyltransferase enzyme also reached a maximum. Finally, dertermination of the serum concentrations of melatonin by use of radioimmunoassay showed an increase during winter. Moreover, our results are in concordance with several biochemical and morphological parameters of the reproductive axis of the male vizcacha, which support the reproductive rhythmicity of this rodent. Thus, our data suggest that the pineal gland and melatonin, which is activated via the sympathetic system, could be involved in the photoperiodically dependent annual reproductive behavior of the vizcacha.

Bilateral Enucleation and Captivity Influence the Reproductive Cycle of Male Viscacha (Lagostomus maximus maximus)

The viscacha (Lagostomus maximus maximus) is a seasonal rodent living in the Southern Hemisphere. The adult males exhibit an annual reproductive cycle characterized by a gonadal regression period during winter. In this study, we investigated the effects of bilateral enucleation and captivity on their annual reproductive cycle. Testicular volume relative to body weight was recorded monthly in intact and bilaterally enucleated animals placed under natural photoperiod, water, and food ad lib. and constant temperature. Testes and accessory organs were evaluated by qualitative and quantitative light microscopic studies. The intact animals showed an annual reproductive cycle with complete gonadal atrophy in the first year. In the second year, testicular regression was observed but attenuated in regard to that recorded in the first winter period, indicating that adaptive changes might be involved. Bilateral enucleation in the viscacha dampened and extended the period of its annual reproductive cycle. The results suggest that both conditions, constant captivity and enucleation, produced stimulatory effects on the reproductive system of this rodent. Furthermore, local control mechanisms might be responsible for the morphological differences observed in testes, epididymis, and seminal vesicles from both groups, which exhibited similar levels of serum testosterone. Finally, an intact retinohypothalamic‐pineal axis and/or photoperiodic input would be necessary to maintain the reproductive cycle amplitude and timing in viscacha.

Lithium effect on testicular tissue and spermatozoa of viscacha (Lagostomus maximus maximus). A comparative study with rats.

We investigated the effect of lithium chloride administration (Sigma): 1 mmol/kg b.w. i.p./day for 35 days on the testes and sperm of viscacha (Lagostomus maximus maximus), a nocturnal rodent found only in the pampas of Argentina. The histological study showed that hypospermatogenesis and the sperm number per mL decreased markedly in comparison with the controls (treatment group: 315 x 10(6) +/- 77 x 10(6); control group: 693 x 10(6) +/- 39 x 10(6), Means +/- SEM, Students t-test: p < 0.05). The sperm motility and viability were also affected. Under the same treatment, the testicular tissue and the sperm of rats were not damaged. Moreover, lithium induced these changes when the plasm levels were within the therapeutic range in humans. Our results provide evidence for the claim that viscacha testes and sperm react very sensitively to low doses of lithium, whereas these concentrations do not produce damage in rats.

Daily morphological variations in the viscacha (Lagostomus maximus maximus) retina. Probable local modulatory action of melatonin

Given that the local melatonin levels exhibit rhythmic daily changes in the retina of the viscacha, we considered it important to study the likely daily variations in morphology and specific 2‐[125I]‐iodomelatonin binding in retinas from this rodent and to correlate these putative changes with local indole levels. Adult animals of both sexes were captured in their habitat and were kept under a natural photoperiod. For light and electron microscopic studies the viscachas were sacrificed by decapitation at 08:00, 16:00, and 24:00 hr. A computer‐assisted image analysis system was used to measure the thickness of the complete retina, the photoreceptor layer, the rod outer and inner segments, and the outer nuclear layer. The daily variation in 2‐[125I]‐iodomelatonin binding sites was followed during a 24‐hr light‐dark cycle, the animals being sacrificed at six time points. The parameters studied showed significant variations throughout the 24‐hr period. Maximal specific binding, lysosomal content in the pigment epithelium, and photoreceptor layer outer segment thicknesses were observed at 24:00 hr. Close contact between photoreceptor membranes and microvilli of the pigment epithelium was observed at 08:00 and 16:00 hr. Moreover, the minimal outer segment thickness at 16:00 hr was accompanied by a scarcity of dense bodies, such as lysosomes, a maximum dispersion of melanin pigment granules, and a minimum density of radioligand binding sites. Therefore, in the retina of the viscacha, we suggest that the interaction between melatonin and specific sites could be one of the factors or causes that participate in the regulation of the daily morphological changes observed in viscacha. Anat Rec 266:198–206, 2002.

Effect of Lithium on the Rhythms of Melatonin in the Pineal Gland, Serum and Retina of Viscacha (Lagostomus maximus maximus)

The aim of this work was to describe the pineal, serum and retinal melatonin daily rhythms in viscacha (Lagostomus maximus maximus), a nocturnal and subterranean rodent. A daily rhythm with a nocturnal peak and an increase during the light phase was found in all cases, with the seric rhythm being delayed with respect to the pineal one. Chronic lithium administration to viscachas suppressed the diurnal increase of pineal melatonin, abolished the retinal rhythm and had no effect on seric melatonin.

Daily rhythms of norepinephrine, β1-adrenoceptor mRNA, serotonin, arylalkylamine N-acetyltransferase mRNA, arylalkylamine N-acetyltransferase and hydroxyindol-O-methyltransferase activities, and melatonin in the pineal gland of viscacha

Abstract We described the daily rhythms of parameters involved in melatonin production in the pineal of viscacha. Norepinephrine showed the highest levels at night. A peak in β1-adrenoceptor mRNA was observed at the end of the light phase. Serotonin exhibited higher values during the light period. AA-NAT mRNA showed the highest expression at Zeitgeber time 0 (ZT0), reaching a minimum at night. AA-NAT activity and melatonin content exhibited a nocturnal peak at ZT16, a lower diurnal increase during the light period and minimal level at ZT8. HIOMT activity showed a peak at ZT16. The daily pattern in norepinephrine content was similar to that of AA-NAT activity and melatonin levels. Our data suggest that the melatonin synthesis in viscacha might be activated via adrenergic induction; the differences observed in the daily rhythms of AA-NAT activity and its mRNA suggest that post-transcriptional mechanisms may be involved, and other Zeitgeber and other mechanisms might also control melatonin production.

Differential response of pineal microglia to surgical versus pharmacological stimuli

Microglial cells are one of the interstitial elements of the pineal gland (PG). We recently reported the pattern of microglia colonization and activation, and microglia‐Pax6+ cell interactions during normal pineal ontogeny. Here, we describe the dynamics of microglia‐Pax6+ cell associations and interactions after surgical or pharmacological manipulation. In adult rats, the superior cervical ganglia (SCG) were exposed, and either bilaterally excised (SCGx) or decentralized (SCGd). In the SCGx PGs, the density of Iba1+ microglia increased after surgery and returned to sham baseline levels 13 days later. Pineal microglia also responded to SCGd, a more subtle denervation. The number of clustered Iba1+/PCNA+/ED1+ microglia was higher 4 days after both surgeries compared to the sham‐operated group. However, the number of Pax6+/PCNA− cells and the percentage of Pax6+ cells contacted by and/or phagocytosed by microglia increased significantly only after SCGx. Separate groups of rats were treated with either bacterial lipopolysaccharides (LPS) or doxycycline (DOX) to activate or inhibit pineal microglia, respectively. Peripheral LPS administration caused an increase in the number of clustered Iba1+/PCNA+/ED1+ microglial cells, and in the percentage of Pax6+ cells associated with and/or engulfed by microglia. In the LPS‐treated PGs, we also noted an increase in the number of PCNA+ cells that were Iba1− within the microglial cell clusters. The density of Pax6+ cells did not change after LPS treatment. DOX administration did not influence the parameters analyzed. These data suggest that pineal microglia are highly receptive cells capable of rapidly responding in a differential manner to surgical and pharmacological stimuli.

Effect of lithium on the melatonin production in the pineal gland of viscacha

Abstract Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β1-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.