Ultrastructural and biochemical seasonal changes in epididymal corpus and cauda of viscacha (Lagostomus maximus maximus)

The reproductive and adaptative behavior of wild rodents is synchronized primarily by the photoperiod. The viscacha, a South American rodent of nocturnal habits and seasonal reproduction is photoperiod‐dependent and its reproductive behavior is regulated by the retinohypothalamic‐pituitary pineal axis. Adult males exhibit an annual reproductive cycle with periods of maximum gonadal activity (summer‐early autumn) and gonadal regression (winter). The corpus and the cauda, the most sensitive segments of the epididymis to changes induced by the photoperiod, were analyzed using electron microscopy and enzymatic biochemistry. During gonadal regression, principal and clear cells showed signs of involution with respect to the activity period. These were characterized by more irregular nuclei, smaller cytoplasms, large vacuoles, altered mitochondria, and glycogen deposits. All cellular populations of the epididymal epithelium in regression presented abundant lysosome‐like dense bodies during the active period. In addition, we measured the activity of four acid glycosidases in the cauda epididymis along the reproductive cycle. N‐acetyl‐β‐D‐glucosaminidase (NAG), an enzyme that degrades endocytosed substances from the epididymal lumen, increased significantly during gonadal regression relative to the active period. These results demonstrate that the viscacha epididymis exhibits significant ultrastructural and biochemical changes during the reproductive cycle. We demonstrate that during regression, melatonin secretion in viscacha increases. This study shows that the epididymal epithelium is reduced. Thus, we postulate that the changes observed in the epididymis are modulated by pineal melatonin. Despite these changes, the epididymis might maintain a microenvironment suitable for the survival of stored spermatozoa. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Seasonal variations in the testis and epididymis of vizcacha (Lagostomus maximus maximus).

Seasonal changes in reproductive activity in the adult male vizcacha (Lagostomus maximus maximus), a South American rodent, were investigated. Monthly, for 2 yr, the animals were killed and decapitated during the night near their burrows in the vicinity of San Luis, Argentina. The testes, epididymides, and pineal glands were removed and used for biochemical and structural studies. Significant changes associated with seasonal cycles were found. 1) In July-August (winter in South America), a short hibernal period of sexual quiescence, decline in testicular and epididymal weights, arrest of spermatogenesis, and decrease of serum testosterone were observed. The gonads regressed during this period, with regression most pronounced in August. 2) During September-November (spring), a recovery period--without arrest of spermatogenesis--was observed, with significant expression of gonadal activity during April-May (autumn). In this season, gonadal weight was increased and spermatogenesis was complete. These results indicate an increase in sexual activity as well as in the ability to secrete testosterone. A gradual reduction of testicular activity appeared in June-July (early winter). Conversely, in this period, the pineal hydroxyindole-O-methyl transferase activity decreased in contrast to the highest values observed in winter. Our findings indicate that the male adult vizcacha under natural conditions exhibits an annual reproductive cycle. A possible relationship between increased pineal activity and gonadal regression is also suggested.     

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.     

Seasonal variations of gonadotropins in the pars distalis male viscacha pituitary. Effect of chronic melatonin treatment

The gonadotropes, LH and FSH cells, were immunohistochemically identified in the pituitary pars distalis of the adult male viscacha (Lagostomus maximus maximus) using specific antibodies against hLHbeta and hFSHbeta with the streptavidin-biotin-peroxidase complex. The distribution, size and percentage immunopositive area of these cells were analyzed by image analysis in viscachas captured during the annual reproductive cycle and after the chronic administration of melatonin. The LHbeta and FSHbeta cells showed seasonal changes in the distribution, size and percentage immunopositive area. The LHbeta cells were found widely distributed throughout the pars distalis during the reproductive period, and they were found in the ventro-medial region in the pars distalis during the gonadal regression and gonadal recovery periods. The LHbeta cells reached the largest size and immunopositive area during the reproductive period and the smallest size and immunopositive area during the gonadal regression period. The FSHbeta cells were found in the ventro-medial region during reproductive and gonadal regression periods. The FSHbeta cells were found widely distributed throughout the pars distalis during the gonadal recovery period when they showed the maximum percentage immunopositive area. A decrease in the size of LHbeta and FSHbeta cells was observed after the chronic administration of melatonin. Moreover, it produces a decrease in the immunopositive area occupied by the LHbeta cells but not in the immunopositive area occupied by the FSHbeta cells. Our results show great activity of LHbeta and FSHbeta cells in different moments of the annual reproductive cycle demonstrating that these cells do not secrete in parallel. Moreover, melatonin acts differentially on the activity of the gonadotrope cells.     

Evidence of an annual rhythm in a small proportion of Siberian hamsters exposed to chronic short days

Siberian hamsters are photoperiodic seasonally breeding rodents. To date, there has been no evidence that Siberian hamsters exhibit an annual rhythm in reproductive, thermoregulatory, molt, or body mass changes. However, given that the termination of their winter cycle is under endogenous control, the authors thought it possible that under particular conditions, other aspects of their seasonal cycle may be endogenously mediated. Consequently, the authors monitored the reproductive condition, body mass, and molt of hamsters chronically exposed to short days (LD 9:15) for up to 2 years. All animals were taken from previous experiments and had exhibited gonadal regression, followed by refractoriness to shorts days and spontaneous gonadal growth, as well as a complete cycle of body mass and molt changes. Although some hamsters died during the study, the authors continued to monitor some animals for up to 106 weeks of short-day exposure. Of the 57 animals monitored, 4 (7.02%) exhibited a second cycle of reproductive regression and recrudescence. Furthermore, the timing of the second regression was similar in the 4 animals, occurring about 1 year after the initial short-day exposure (50.5 +/- 1.71 weeks). However, the timing of the second bout of recrudescence was more variable, occurring at about week 80 (79.5 +/- 8.01 weeks). Hamsters exhibited regressed gonads for about 7 months (29.0 +/- 8.02 weeks). Although the body mass of the 4 hamsters declined at the time of the second cycle of gonadal regression, it never recovered. No hamster exhibited a second molt cycle. These observations indicate that a small percentage of Siberian hamsters chronically exposed to short days can exhibit an annual cycle of reproduction.     

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, Student's 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.     

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.     

Photoperiod, follicle-stimulating hormone receptors, and testicular function in vizcacha (Lagostomus maximus maximus)

In the present work we investigated the presence of testosterone in serum and follicle-stimulating hormone (FSH) receptors in testes of the vizcacha (Lagostomus maximus maximus), a South American rodent. We also investigated the effect of constant light on both parameters. The control group consisted of vizcachas caught in their natural habitat and maintained under continuous darkness; the experimental group consisted of animals maintained under constant light (1076 lx) for 8 days. The results revealed a significant decrease in serum testosterone and FSH receptors when the animals were maintained under constant light, as compared to the control group. Androstenedione was elevated in the serum obtained from the experimental group. It is postulated that the pineal gland may regulate testosterone secretion through FSH receptors and through an enzymatic blockade in the steroidogenic pathway; this supposition, however, remains to be proved.     

Morphological characteristics of the uterus and uterine cervix of the plains viscacha (Lagostomus maximus)

The plains viscacha is a hystricognathi rodent with special reproductive characteristics. Despite poly-ovulation of 800 oocytes, it generates a high embryonic mortality since from 10 to 12 implantations only one or two offspring are born. The present work analyses the morphological, morphometrical, histochemical and lectinhistochemical characteristics of the uterus and uterine cervix of pregnant and non-pregnant viscacha. Anatomically, the uterus has two horns each one continued with a short cervix devoid of glands. The structure of the uterus is like that of other species; however, the proportion and size of its glands varies according to the physiological state. In viscacha, there is no uterine body and cervices cannot be differentiated externally. The formation of two sack bottoms is determined by the presence of a middle raphe in the cranial portion of the vagina. The so-described anatomy is different from that described in other hystricognathi such as guinea pig and coypu. The cervix presents two microscopically differentiable regions: the endocervix and the ectocervix lined by epithelia with different characteristics. The general characteristics of the uterus of L. maximus do not show specific differences with those of other mammals that might explain its peculiar gestation.     

Seasonal regulation of reproduction: altered role of melatonin under naturalistic conditions in hamsters

The seasonal reproductive cycle of photoperiodic rodents is conceptualized as a series of discrete melatonin-dependent neuroendocrine transitions. Least understood is the springtime restoration of responsiveness to winter-like melatonin signals (breaking of refractoriness) that enables animals to once again respond appropriately to winter photoperiods the following year. This has been posited to require many weeks of long days based on studies employing static photoperiods instead of the annual pattern of continually changing photoperiods under which these mechanisms evolved. Maintaining Siberian hamsters under simulated natural photoperiods, we demonstrate that winter refractoriness is broken within six weeks after the spring equinox. We then test whether a history of natural photoperiod exposure can eliminate the requirement for long-day melatonin signalling. Hamsters pinealectomized at the spring equinox and challenged 10 weeks later with winter melatonin infusions exhibited gonadal regression, indicating that refractoriness was broken. A photostimulatory effect on body weight is first observed in the last four weeks of winter. Thus, the seasonal transition to the summer photosensitive phenotype is triggered prior to the equinox without exposure to long days and is thereafter melatonin-independent. Distinctions between photoperiodic and circannual seasonal organization erode with the incorporation in the laboratory of ecologically relevant day length conditions.     

Seasonal variations in plasma luteinizing hormone levels of gonadectomized male ground squirrels (Spermophilus lateralis)

Plasma luteinizing hormone (LH) levels were determined at monthly intervals in intact and orchidectomized ground squirrels maintained in a constant 14L:10D photoperiod at a temperature of 23 +/- 2 degrees C. Animals were orchidectomized or sham operated at different ages and at different stages of the annual reproductive cycle. LH levels were elevated in orchidectomized as compared to intact males within 1 month, and often within 48 h, after orchidectomies performed either before or after the normal breeding season. LH levels tended to remain chronically elevated in long-term orchidectomized males. Although negative feedback regulation of LH by the testes was evident at most stages of the reproductive cycle, effects of orchidectomy on LH were most pronounced during the nonbreeding season when plasma LH titers of intact males were at their annual nadirs, and less pronounced or absent during the breeding season when levels were normally elevated in intact males. For most of the year male gonadal hormones exert negative feedback on LH release. We hypothesize that under the influence of a circannual timing mechanism, sensitivity to steroid feedback is reduced as the breeding season approaches and LH secretion increases despite the attendant rise in plasma androgen levels. These results contrast with data for female squirrels in which there is a seasonally restricted and characteristically brief elevation of plasma LH after ovariectomy; in females, but not in males, onset and termination of LH secretion appear to be regulated by a circannual clock that functions independently of gonadal secretions.     

Photoperiod and temperature interact to affect the GnRH neuronal system of male prairie voles (Microtus ochrogaster)

Individuals of numerous species limit energy expenditure during winter by inhibiting reproduction and other nonessential functions. To time these adaptations appropriately with the annual cycle, animals rely on environmental cues that predict, well in advance, the onset of winter. The most commonly studied environmental factor that animals use to time reproduction is photoperiod. Rodents housed in short photoperiods in the laboratory or in naturally declining day lengths exhibit pronounced alterations in reproductive function concomitant with alterations in the hypothalamic gonadotropin-releasing hormone neuronal system. Because animals in their natural environment use factors in addition to photoperiod to time reproduction, the present study sought to determine the independent effects of photoperiod and temperature, as well as the interaction between these factors, on reproductive parameters and the GnRH neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 10 weeks. Animals in each photoperiod were further subdivided into groups housed in either mild (i.e., 20 degrees C) or low (i.e., 8 degrees C) temperatures. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods and long-day voles housed in low temperatures (and maintained large gonads) exhibit higher GnRH-immunoreactive (GnRH-ir) neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to all other groups. In addition, voles that underwent gonadal regression in response to both short days and low temperatures did not exhibit an increase in GnRH-ir neuron numbers compared to long-day, mild-temperature controls. These data suggest that photoperiod and temperature interact to influence reproductive function potentially by alterations of the GnRH neuronal system.     

An atypical reproductive cycle in a common viviparous Asia Agamid Phrynocephalus vlangalii

Viviparous lizards living in cold climate of high altitude often exhibit atypical reproductive cycles, in which mating and fertilization occur synchronously and annually with parturition occurring at the end of the year. Nevertheless, detailed case studies on atypical reproductive cycles are few. Using anatomical data combined with behavioral observations, we examined the reproductive cycle of a common Asian agamid, Phrynocephalus vlangalii, from a high‐elevation area in Sichuan, China. Male spermiation of P. vlangalii occurred in May, and spermatogenesis began in June and reached a maximum in October. For females, ovulation and fertilization occurred in May, and females developed gestation and pregnancy in 3 months from June to August, without vitellogenesis during this period. Females gave birth synchronously in late August, then vitellogenesis began and lasted until May of the next year. All adult males and females were synchronized in the same reproductive condition each month. The synchronous and annual reproductive cycle of P. vlangalii clearly represents an atypical cycle. The male courtship and mating behaviors were concordant with gonadal cycle and mainly happened in May and June. Despite the short growth period for neonates, they had a high over‐winter survival rate of 84.4%, suggesting that autumn parturition did not generate high costs to this reproductive cycle. We propose that the high over‐winter survival rate of neonates is likely linked with female delayed sexual maturity, female asynchronous vitellogenesis and gestation, large relative clutch mass (RCM), and adult‐offspring burrow sharing behavior during hibernation.     

Delayed effect of pinealectomy on hibernation of the golden-mantled ground squirrel

Pinealectomy or radical sham pinealectomy were performed on adult golden-mantled ground squirrels,Spermophilus (=Citellus) lateralis, approximately 1 month prior to the date of normal winter emergence. The first hibernatory period and subsequent active season were not different in either of the operated groups from intact animals. However, although the initiation of the second hibernatory period was not affected in the pinealectomized animals, this group failed to show the progressive increase in the length of heterothermic bouts that is characteristic of normal hibernation. Also, terminal arousal occurred approximately 6 weeks earlier in the second year after pinealectomy. Male squirrels showed a corresponding time compression in their annual gonadal cycle, as was assessed by testicular state.These results suggest that the pineal gland of the golden-mantled ground squirrel is involved in the expression of the annual hibernatory cycle. In the absence of the pineal gland the adult of this species is unable to sustain the normal depth and duration of hibernation in the second over-wintering period following pinealectomy.We have carried out additional experiments with young, laboratory-bornS. lateralis and with field-caught, adultS. richardsonii. The results of these studies also are described in this paper.Presented at the Ninth International Congress of Biometeorology, 23 Sept – 1 Oct 1981, Osnabrück and Hohenheim, FRG.     

Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides)

The objectives of this study were to (1) assess year-round behaviors and activity patterns of captive raccoon dogs (Nyctereutes procyonoides) and (2) characterize the species' reproductive endocrinology. Behaviors and activity patterns of 12 (5.7) animals were recorded over a 1-year period. During that time, fecal samples were collected 2-7 times/week from 16 (7.9) individuals (six of these were included in the behavioral study) for the analysis of testosterone, progesterone and estrogen metabolite concentrations. Activity pattern and excretion of gonadal steroids followed a seasonal pattern. Specifically, dogs were cathemeral in summer, and primarily nocturnal in winter. In the males, testosterone concentrations were at baseline from April through September, began to rise in October and reached peak concentrations in February (P<0.05). In the females, elevated estrogen (P<0.05) was observed in March followed by an increase in progestagen concentrations from March through May (P<0.05) in both pregnant and pseudopregnant animals. Gender significantly influenced monthly testosterone/estrogen ratio (P<0.01); values were higher in males than in females throughout the year with overall percentage of overlapping values between males and females being 28%. In summary, this study characterized cirannual fluctuations in behaviors and gonadal steroid metabolites in the raccoon dog maintained in captivity. Because there is no obvious sexual dimorphism, the differences in testosterone/estrogen ratio may be useful for gender differentiation (72% accuracy), especially among individuals living in the wild.     

Annual reproductive activity of Caiman crocodilus fuscus in captivity

We describe the gonadal and abdominal fat storage cycles for a population of Caiman crocodilus fuscus kept in captivity at a breeding farm. The reproductive cycles of males and females are clearly seasonal. Vitellogenic follicles were observed in females year‐round; however, the follicular growth that produces the preovulatory follicles for the breeding season occurs in November–May (most of the dry season to the beginning of the rainy season). These months correspond with the presence of physiologically reproductive males. Oviposition takes place during the wet season (April–August) and the births occur at the end of this season (September–November). Fat storage was not related to reproductive activity, and did not vary by month or between sexes. Although environmental and hormonal conditions have been improved for these captive animals, they maintain a strongly seasonal reproductive cycle, historically fixed to oviposit in the most favorable period for nest construction and hatching during the rainy season. Zoo Biol 22:121–133, 2003. © 2003 Wiley‐Liss, Inc.     

Gonadal responses to food restriction in intact and pinealectomized male golden hamsters

Male golden hamsters underfed for 11-16 weeks showed a 30% decrease in body weight accompanied by significant testicular regression. Pinealectomy did not prevent underfeeding-induced gonadal regression. Resumption of ad-libitum feeding restored body weights to near control levels and permitted testicular redevelopment in animals with previously regressed tests. These data indicate that chronic food restriction can affect the reproductive status of hamsters and that this effect does not depend on an intact pineal gland. Body weight changes may serve as a secondary proximate cue in addition to photoperiod for timing the hamster seasonal breeding cycle.     

Biochemical and energy dynamics throughout the reproductive cycle of the striped venus Chamelea gallina (Mollusca,Bivalvia)

The striped venus Chamelea gallina is an important commercial bivalve species in Europe. However, large inter-annual fluctuations in stock abundance and periodic recruitment failure threaten the biological and economic sustainability of this fishery. This study aimed to improve the knowledge of the reproductive cycle and reproductive strategies of this species from the Algarve coast (southern Portugal) in order to contribute to the establishment of management measures and to assess its potential for aquaculture. The reproductive cycle of C. gallina followed a seasonal cycle, significantly influenced by sea surface temperature and food availability. Gametogenesis took place in winter, coinciding with the phytoplankton bloom. Spawning occurred during summer, followed by a short period of sexual inactivity in autumn. Condition index did not reflect the reproductive cycle of C. gallina and generally, followed the same trend of chlorophyll a. Glycogen was positively correlated with gonadal index and chlorophyll a. High total lipid values were recorded throughout gonad ripeness and spawning, but decreased at the end of the spawning and in the rest period. The extended spawning period of C. gallina will allow larvae to be obtained for much of the year by artificial spawning of wild broodstock.