Prolactin Is a Strong Candidate for the Regulation of Luteal Steroidogenesis in Vizcachas (Lagostomus maximus).

Prolactin (PRL) is essential for the maintenance of the corpora lutea and the production of progesterone (P4) during gestation of mice and rats, which makes it a key factor for their successful reproduction. Unlike these rodents and the vast majority of mammals, female vizcachas (Lagostomus maximus) have a peculiar reproductive biology characterized by an ovulatory event during pregnancy that generates secondary corpora lutea with a consequent increment of the circulating P4. We found that, although the expression of pituitary PRL increased steadily during pregnancy, its ovarian receptor (PRLR) reached its maximum in midpregnancy and drastically decreased at term pregnancy. The luteinizing hormone receptor (LHR) exhibited a similar profile than PRLR. Maximum P4 and LH blood levels were recorded at midpregnancy as well. Remarkably, the P4-sinthesizing enzyme 3ß-HSD accompanied the expression pattern of PRLR/LHR throughout gestation. Instead, the luteolytic enzyme 20α-HSD showed low expression at early and midpregnancy, but reached its maximum at the end of gestation, when PRLR/LHR/3ß-HSD expressions and circulating P4 were minimal. In conclusion, both the PRLR and LHR expressions in the ovary would define the success of gestation in vizcachas by modulating the levels of 20α-HSD and 3ß-HSD, which ultimately determine the level of serum P4 throughout gestation.

Quantification of healthy and atretic germ cells and follicles in the developing and post-natal ovary of the South American plains vizcacha, Lagostomus maximus: evidence of continuous rise of the germinal reserve.

The female germ line in mammals is subjected to massive cell death that eliminates 60-85% of the germinal reserve by birth and continues from birth to adulthood until the exhaustion of the germinal pool. Germ cell demise occurs mainly through apoptosis by means of a biased expression in favour of pro-apoptotic members of the BCL2 gene family. By contrast, the South American plains vizcacha, Lagostomus maximus, exhibits sustained expression of the anti-apoptotic BCL2 gene throughout gestation and a low incidence of germ cell apoptosis. This led to the proposal that, in the absence of death mechanisms other than apoptosis, the female germ line should increase continuously from foetal life until after birth. In this study, we quantified all healthy germ cells and follicles in the ovaries of L. maximus from early foetal life to day 60 after birth using unbiased stereological methods and detected apoptosis by labelling with TUNEL assay. The healthy germ cell population increased continuously from early-developing ovary reaching a 50 times higher population number by the end of gestation. TUNEL-positive germ cells were <0.5% of the germ cell number, except at mid-gestation (3.62%). Mitotic proliferation, entrance into prophase I stage and primordial follicle formation occurred as overlapping processes from early pregnancy to birth. Germ cell number remained constant in early post-natal life, but a remnant population of non-follicular VASA- and PCNA-positive germ cells still persisted at post-natal day 60. L. maximus is the first mammal so far described in which female germ line develops in the absence of constitutive massive germ cell elimination.

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.

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species

The developing ovary of the South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia): massive proliferation with no sign of apoptosis-mediated germ cell attrition.

Apoptosis-dependent massive germ cell death is considered a constitutive trait of the developing mammalian ovary that eliminates 65-85% of the germinal tissue depending on the species. After birth and during adult lifetime, apoptotic activity moves from the germ cell proper to the somatic compartment, decimating germ cells through follicular atresia until the oocyte reserve is exhausted. In contrast, the South American rodent Lagostomus maximus shows suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation, which finally exhausts the oocyte pool. The absence of follicular atresia in adult L. maximus might arise from a failure to move apoptosis from the germinal stratum to the somatic compartment after birth or being a constitutive trait of the ovarian tissue with no massive germ cell degeneration in the developing ovary. We tested these possibilities by analysing oogenesis, expression of germ cell-specific VASA protein, apoptotic proteins BCL2 and BAX, and DNA fragmentation by TUNEL assay in the developing ovary of L. maximus. Immunolabelling for VASA revealed a massive and widespread colonisation of the ovary and proliferation of germ cells organised in nests that disappeared at late development when folliculogenesis began. No sign of germ cell attrition was found at any time point. BCL2 remained positive throughout oogenesis, whereas BAX was slightly detected in early development. TUNEL assay was conspicuously negative throughout the development. These results advocate for an unrestricted proliferation of germ cells, without apoptosis-driven elimination, as a constitutive trait of L. maximus ovary as opposed to what is normally found in the developing mammalian ovary.

Fast-developing preimplantation embryo progeny from heterogametic females in mammals.

Karyotyping and cell number estimates in preimplantation embryos from heterogametic (XY*) and homogametic (XX) females of the field mouse Akodon azarae were studied to determine whether XX-XY-XY* differences exist in the rate of preimplantation development. At the morula stage, XY embryos from heterogametic mothers had twice the mean number of cells compared with XX embryos. However, this difference in cell numbers was not seen between XX and XY embryos from homogametic mothers. In this case, mean cell numbers were similar despite embryos being XX or XY. Furthermore, the mean cell number for XX and XY morulae from homogametic females was comparable to that for XX embryos from heterogametic females. It is concluded that XY* embryos (which will develop into heterogametic females) show an accelerated rate of preimplantation development.

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure.

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae).

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.

Superovulation in vesper mice, Calomys laucha--an important biomedical model for hantavirus and arenavirus (Rodentia-Sigmodontinae).

Sigmodontine rodents are poorly studied and have not received much attention as a reproductive model. Renewed interest in the South American rodents has been stimulated by their link to endemic diseases that are transmitted to man. Calomys laucha acts as a reservoir of two dangerous viruses: an arenavirus named 'Junin virus', the aetiological agent of Argentinian haemorrhagic fever, and the hantavirus, both of which constitute serious sanitary problems. The aim of this study was to establish suitable conditions to superovulate the vesper mouse, Calomys laucha. We examined the hormonal doses, the time interval between hormones, the time-course of ovulation, and the effect of female age on the response to exogenous hormone administration. Female mice were injected with 5-5, 8-8 or 12-15 IU of PMSG/hCG, 48 h apart, at different age intervals (from 30 to > 120 days old). The best superovulation rate was obtained with 8-8 IU PMSG/hCG. Ovulation started about 10 h post-hCG and was completed during the next 4-5 h, and was achieved irrespectively from the oestrus cycle stage. The number of oocytes was influenced by the age of the females. The youngest females had only a superovulatory response. Females older than 61 days showed both ovulatory and superovulatory responses, although 91-120-day-old females had a high ovulatory response. Most of the oocytes (96.5%) recovered were morphologically normal. The genus Calomys constitutes a reproductive model completely different from conventional laboratory rodents.

Cytogenetics of vesper mice, Calomys (Sigmodontinae): a new karyotype from the Puna region and its implication for chromosomal phylogeny.

The karyotype of specimens identified as Calomys lepidus, trapped at 3600 m above sea level in the Puna region, northwestern Argentina, was studied. All specimens analysed showed a 2n = 44 (NF(a) = 68) asymmetrical karyotype with 13 pairs of metacentric/submetacentric autosomes and 7 pairs of telocentric chromosomes. The X was a medium-sized submetacentric and the Y a small submetacentric chromosome. This karyotype was quite different from that previously described for C. lepidus from Peru (2n = 36, NF(a) = 68). However, both karyotypes may be easily interrelated by means of four centric fusions, and the chromosome complement of Punian C. lepidus fitted into a previously proposed chromosomal phylogeny of the genus. In addition, the spermatozoa of specimens corresponded to a morphological pattern previously described for other species of Calomys.

Offspring sex-ratio and reproductive performance in heterogametic females of the South American field mouse Akodon azarae. Reproduction in heterogametic Akodon azarae females.

We have compared the breeding performance of homogametic (XX) and heterogametic (XY*) females of the South American sigmodontine rodent Akodon azarae under laboratory conditions. XY* females showed an enhanced reproductive performance when compared with normal, XX, females. The XY* females had a longer reproductive lifespan. They started to reproduce early, had more frequent litters, and stopped reproduction later than XX females. Their progeny showed a biased 1:2 male:female sex ratio which may be explained by the early loss of YY* zygotes after fertilization. However, litter size at birth was similar both in XY* and XX females, and no difference in ovulation rate was detected between them. This indicates that an "automatic" rather than an "evolved" reproductive compensation mechanism may be acting in heterogametic females. A separate study has shown that self-synapsis of both the X and Y* chromosomes takes place during meiosis, allowing the oocytes to escape from functional deterioration. It is suggested that self-synapsis and "automatic" reproductive compensation account for the preservation of fertility in heterogametic females in spite of the heteromorphic sex chromosomes and the early embryo loss they experience. However, these mechanisms do not account for the enhancement of reproductive lifespan. The possibility that an intrauterine position phenomenon is acting in A. azarae is discussed.

Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation.

The main role of the ovarian granulosa cells is to nurse the oocyte and to produce estradiol and progesterone upon stimulation by gonadotropins. In fact, follicle-stimulating hormone (FSH) and luteinizing hormone control the expression of several genes during granulosa cell differentiation via cyclic AMP-dependent phosphorylations. Cyclic AMP stimulates transcription of genes that carry the cAMP-responsive element (CRE,5'TGACGTCA3') in their promoters. The fibronectin (FN) gene contains one CRE sequence at position -170. However, gonadotropins and cAMP inhibit FN gene expression in granulosa cells. To study the mechanism of the inhibition we developed a bovine granulosa cell line (BGC-1) that synthesizes estradiol in response to FSH and in which FSH and dibutyryl cAMP specifically decrease FN synthesis and its mRNA levels. The inhibitory effect (a) is not due to an alteration in FN mRNA stability, (b) requires upstream sequences other than CRE, located between positions -510 and -223, that are able to bind granulosa cell nuclear proteins, (c) is entirely dependent on the synthesis of intermediate proteins induced and or phosphorylated by cAMP, and (d) effectively suppresses the CRE-dependent transcriptional activation.

Vertical transmission of Junin virus in experimentally infected adult Calomys musculinus.

The response to infection with Junin virus, wild strain Cba An 9446, and the antenatal and postnatal transmission of the pathogen in its natural host, Calomys musculinus, were studied. Intranasal infection in adult animals (90-120 days) did not produce mortality or illness during the 150-day period of observation. From day 21 to 150 after infection, 50% of the animals showed viral persistence with shedding of virus in both urine and saliva. The remaining half became seropositive, and no infectious virus was recovered from them. Although the virus did not infect fetuses during gestation, 50% of weaned pups nursed by viremic mothers were infected. Neither persistence nor immunologic response altered the reproductive pattern of the animals. The absence of reproductive failure in the infected host and the efficiency of postnatal transfer of Junin virus indicate that vertical transmission could contribute to the viral maintenance over time.

Meiotic behavior of gonosomically variant females of Akodon azarae (Rodentia, Cricetidae).

The meiotic behavior of sex chromosomes has been investigated in variant females of Akodon azarae, both in pachytene oocytes and metaphase I. In somatic cells, these females have a heteromorphic sex pair, in which the minor chromosome has been previously interpreted as a major deletion of the long arm of the X chromosome (dX). After microspreading for synaptonemal complex analysis, pachytene oocytes show two axes of very different lengths (100:17.1), which correspond to the sex chromosomes X and dX. True synapsis is abnormally restricted (43.3%) between these sex chromosomes; on the other hand, self-synapsis of both the X and dX chromosomes is frequent (60%). Single, nonsynapsed axes or axial segments are thickened. Strong chromatin condensation occurs around nonsynapsed axes or axial segments, giving many of these sex pairs an appearance similar to an XY body ("sex vesicle"). The minor gonosome axis differs from that of the Y chromosome of male meiosis, as the former is shorter (relative to the X) and has a different synaptic behavior. In 17 metaphases I from XdX variant females, only heteromorphic, end-to-end joined sex pairs were observed. These variant females differ from the variant females of the wood lemming Myopus schisticolor in several respects, but a similar mechanism seems to be prevalent in other species of the genus Akodon. Self-synapsis of unequal gonosomes in oocytes is assumed as an escape from functional deterioration, following the hypothesis put forward by others.

Is vertical transmission sufficient to maintain Junin virus in nature?

The quantitative contribution of vertical transmission to the prevalence rate of Junin virus infection in subsequent generations of its natural reservoir, Calomys musculinus, was analysed. Data on mortality and reproduction of C. musculinus infected at birth with a wild strain of Junin virus were used to estimate the infection-dependent relative survival rate (beta = 0.4849) and relative fertility of the infected host (alpha = 0.2088). Prevalence rates of infection, obtained by mathematical simulation in optimal conditions of vertical transfer, dropped steadily to zero in a few generations. Vertical transmission was found to be insufficient to overcome the effect of highly depressed survival and fertility of the infected host and maintain a stabilized prevalence of Junin virus infection in successive generations; this suggested that viral maintenance is mainly dependent upon horizontal transmission.

Effect of persistent infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus.

The effect of infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus, was studied. Eighty-five C. musculinus were inoculated intranasally at birth with 100 TCID50 of Cba An 9446 strain of Junin virus and observed for 480 days. No clinical signs of neurologic illness were registered. Infected animals showed an increased mortality rate of up to 70% between days 24-40 post-infection. This period of high mortality was preceded by low weight gain during lactation and registered until 60 days. From day 14 post-infection until day 480, Junin virus was recovered from blood, urine, and oral swab in all animals checked at any time. By day 480 post-infection, 100% of survivors showed widespread viral dissemination in brain, spleen, kidneys, and salivary glands. There was marked reduction in reproductive efficiency among infected animals. Out of 15 mating pairs, 2 (13.3%) littered at least once compared to 60% in the control group. The reduction of fertility and the altered survival rate of Junin virus-infected C. musculinus indicate that vertical transmission mechanisms per se are insufficient to maintain the infection in successive generations in the absence of horizontal transmission.