The ovary of the gestating South American plains vizcacha (Lagostomus maximus): suppressed apoptosis and corpora lutea persistence

The South American plains vizcacha, Lagostomus maximus, displays an exceptional ovulation rate of up to 800 eggs per cycle, the highest rate recorded for a mammal. Massive polyovulation arises from the overexpression of the apoptosis-inhibiting BCL2 gene leading to a suppression of apoptotic pathways responsible for follicular atresia in mammals. We analyzed the ovarian histology, ovarian apoptosis, and apoptosis-related protein expression with special emphasis in corpora lutea throughout the 5-mo-long gestation period, at parturition day and early postpartum, in L. maximus. Corpora lutea were abundant throughout gestation with no sign of structural regression even at the end of gestation. Both immunohistochemistry and Western blot analysis showed strong signals for apoptosis-inhibiting BCL2 protein, whereas the proapoptotic BAX protein was just detected in isolated luteal cells in gestating females and postpartum females. Apoptosis-associated DNA fragmentation detected by TUNEL was very scarce and occasional and correlated with BAX detection in luteal cells. Marked expression of progesterone and alpha-estrogen receptors in luteal cells was found at early, mid-, and late gestation as well as at parturition day and early postpartum samples. Additionally, serum level of progesterone increased markedly to reach maximal values at late gestation and decreasing at parturition to levels found at early gestation, suggesting that corpora lutea remained functional throughout gestation. These results point out that the unusual ovarian environment of L. maximus in which germ cell demise is abolished through antiapoptotic BCL2 gene overexpression also preserves structural integrity and functionality of corpora lutea during the whole gestation. Overexpression of antiapoptotic BCL2 gene may represent a strategy for an essential need of ovary and corpora lutea in order to maintain pregnancy until term.     

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.     

Effects of local extinction of the plains vizcacha (Lagostomus maximus) on vegetation patterns in semi-arid scrub

We studied spatial and temporal effects of local extinction of the plains vizcacha (Lagostomus maximus) on plant communities following widespread, natural extinctions of vizcachas in semi-arid scrub of Argentina. Spatial patterns in vegetation were examined along transects extending outward from active and extinct vizcacha burrow systems. Responses of vegetation to removal of vizcachas were assessed experimentally with exclosures and by documenting vegetation dynamics for 6 years following extinctions. Transect data demonstrated clear spatial patterns in plant cover, particularly an increase in perennial grasses, outward from active vizcacha burrows. These patterns were consistent with predictions based on foraging theory and studies that document grasses as the preferred food of vizcachas. Removal of vizcachas, experimentally and with extinctions, resulted in an immediate increase in perennial and annual forbs indicating that intense herbivory can depress forb cover, as well as grasses. After a 1-year lag following cessation of herbivory, cover of grasses increased. Forbs declined as grasses increased. The long-term effect of extinction of vizcachas was a conversion of colony sites from open patches dominated by forbs to dense bunch grass characteristic of the matrix. Major changes in vegetation occurred within 2–3 years after extinction, resulting in a large pulse of landscape change. However, some species of grasses were uncommon until 5–6 years after the vizcacha extinction. With extinction and colonization, vizcachas generate a dynamic mosaic of patches on the landscape and create temporal, as well as spatial, heterogeneity in semi-arid scrub.     

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.     

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.     

Hydroxyindole-O-methyl transferase (HIOMT) and serotonin in the pineal gland of the vizcacha (Lagostomus maximus maximus)

Pineal glands of vizcachas collected from their natural habitat were studied. Hydroxyindole-O-methyl transferase (HIOMT) activity showed a great individual dispersion. However, the enzyme activity was significantly higher in males than in females. Serotonin was also assayed in the glands. It did not show significant differences between both sexes. The results are discussed in relation to the natural life conditions of this rodent.     

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia)

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.     

Exposure to constant light increases pineal weight and RNA content in the pineal of the vizcacha (Lagostomus maximus maximus)

The following parameters, RNA and protein content, gland weight and nuclear size were studied in pineal glands of adult male vizcachas, maintained in captivity under permanent light (Lot I) and darkness (Lot II). The same determinations were performed in pineals of vizcachas shot at night in their normal habitat (Lot III, Control lot). A significant rise of gland weight and RNA content was observed in Lot I (with respect to the Control lot), while nuclear size and protein content were unchanged. The only significative differences between Lots I and II were also found in the pineal weight and RNA content. These results indicate that exposure of vizcachas to permanent light for 10 days does not produce inhibition of the parameters under study as occurs in other species. Apparently, the light acts as a stressor in these nocturnal mammals.     

Histological and histochemical study of the duodenum of the plains viscacha (Lagostomus maximus) at different stages of its ontogenetic development

The general objective of this study focused on the duodenal histological and histochemical analysis of fetuses and adults of plains viscacha (Lagostomus maximus) from the Buenos Aires province (Argentina). Histological techniques, histochemical procedures for localizing and characterizing glycoproteins (GPs) and lectin histochemical techniques for the identification of specific sugar residues were used. The duodenal structure of all age groups here considered was typical of mammals. We identified a proliferation phase and an epithelial morphogenesis in mid‐gestational fetuses, an intermediate period of cell differentiation in at‐term fetuses and a posterior stage of physiological maturation in adults. According to histochemistry, the diverse GPs elaborated and secreted into the duodenum show a high degree of histochemical complexity related to the multiple functions of mucus in the digestive tract. The GPs in the duodenum of L. maximus and their glycosylation patterns varied according to the animal's age and the developmental state of the organ.     

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.     

Distribution of lithium in different CNS areas and other tissues of adult male and female vizcacha (Lagostomus maximus maximus).

In a previous study (1) we demonstrated that lithium administration (1.0 mmol/kg b.wt., per day for 4 weeks) in intact vizcacha (Lagostomus maximus maximus) leads to significant histological alterations in the kidneys, ovarie and testicles, while these three tissues were not damaged in rats. Male vizcachas died within 4 days when administered LiCl 3 mmol/kg b.wt., while females were not affected. The lithium renal clearance presented no changes in either males or females. The 1.0 mmol/kg b.wt. dose was used in the experiments (2). In this study we examined the distribution of lithium in various tissues of male and female vizcacha (Lagostomus maximus maximus) administered LiCl by injection (1 mmol/kg b.wt.) for one day (Group I) and thirty days (Group II). Blood sample was obtained after 24 hours (Group I) and 30 days (Group II). The tissues investigated were: pituitary, hypothalamus, cerebral cortex, cerebellum, corpus callous, small and large intestine, kidney and suprarenal. The concentration of lithium in tissues and serum was determined by atomic absortion spectrometry (3,4). In Group I a significant lithium concentration increment (mumol/g of tissue) was observed in all the tissues of male vizcachas as compared to female vizcacha. A similar distribution was obtained in animals treated for 30 days. In the pituitary, however this difference between males and females was not significant. The male lithium serum levels were significantly higher than those of female animals. In conclusion, we suggest that the particular structure of the cell membrane (e.g., number and characteristic of sodium channels) of each tissue and/or the intracellular mechanisms of transport, elimination and metabolism might explain the unequal lithium distribution and the difference recovery from the damage produced. The results suggest that the vizcacha could be a useful model for the study of lithium toxicity.     

Mammalian gonadotropin-releasing hormone (GnRH) receptor subtypes

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that governs gonadotropin secretion through interaction with its seven transmembrane (7TM), G protein-coupled receptor (GPCR) expressed by anterior pituitary cells. A second decapeptide, GnRH II, originally discovered in the chicken hypothalamus was recently reported to be expressed in the mammalian hypothalamus as well. A search of the recently-sequenced human genome identified a 7TM/GPCR on chromosome 1 that exhibited a higher identity with non-mammalian vertebrate GnRH II receptors (55%) than with the human GnRH I receptor (39%). Molecular cloning and nucleotide sequencing of this putative GnRH II receptor cDNA from monkey pituitary gland revealed a 379 amino acid receptor that, unlike the GnRH I receptor, possessed a C-terminal tail. Heterologous expression and functional testing of the receptor in COS-1 cells confirmed its identity as a GnRH II receptor: measurement of 3H-inositol phosphate accumulation revealed EC(50)s for GnRH II of 0.86 nM and for GnRH I of 337 nM. Ubiquitous tissue expression of GnRH II receptor mRNA was observed using a human tissue RNA expression array and a 32P-labeled antisense riboprobe representing the 7TM region of human GnRH II receptor cDNA. As predicted by the presence of its C-terminal tail, the GnRH II receptor was desensitized by GnRH II treatment whereas the naturally tail-less GnRH I receptor was not desensitized by GnRH I. Pharmacological analysis of the GnRH II receptor revealed that GnRH I 'superagonists' were more potent than GnRH I but less potent than GnRH II. Numerous GnRH I antagonists showed neither antagonistic nor agonistic activity with the GnRH II receptor. The functions of the GnRH II receptor are unknown but may include regulation of gonadotropin secretion, female sexual behavior, or tumor cell growth.     

Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer

The expression of GnRH (GnRH-I, LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the ovary. The proliferation of human ovarian cancer cell lines is time- and dose-dependently reduced by GnRH and its superagonistic analogs. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in these cancer cells. The GnRH receptor rather interacts with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in downregulation of cancer cell proliferation. In addition GnRH activates nucleus factor κB (NFκB) and protects the cancer cells from apoptosis. Furthermore GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (JNK/AP-1) pathway independent of the known AP-1 activators, protein kinase (PKC) or mitogen activated protein kinase (MAPK/ERK).