Testing the Adaptive Sterilization Hypothesis in mice inoculated with Chlamydia muridarum.

The "adaptive sterilization hypothesis" argues that the tendency of sexually transmitted infections (STIs) to cause infertility likely reflects an evolutionary adaptation of these pathogens. For example, some STIs can lead to bilateral occlusions of the oviducts and sterile matings. Cycling females that do not spend time gestating and lactating are ready to mate sooner than fertile females, and therefore, likely to mate more frequently and possibly more promiscuously. These sexual activities are associated with enhanced transmissibility of STIs, and tubal occlusion is a proximate mechanism by which STIs can increase fitness. Our principal objectives were to determine whether female mice inoculated with Chlamydia muridarum mate more frequently than mice inoculated with sterile saline and to test the hypothesis that tubal occlusion following C. muridarum infection modulates mating behavior in a manner that might increase transmissibility of Chlamydia. Similar to C. trachomatis infections in human females, C. muridarum can ascend the reproductive tract of mice, damage and occlude the oviducts, and cause infertility. However, ovarian function and mating activity are maintained following tubal occlusion. Twenty C57Bl/6 mice with regular estrous cycles were given intra-vaginal inocula of C. muridarum and 32 days later paired with a male for 90 days. Nine saline-treated females served as controls. Three Chlamydia-inoculated females were rendered infertile due to bilateral oviductal damage and mated 8 (±0.0) times. Control females mated on average 4.6 (±0.3) times, and 17 Chlamydia-inoculated fertile females, including six females with only a single oviduct occluded, mated on average 4.7 (±0.2) times. Chlamydia-inoculated fertile females with unilateral oviductal damage had significantly smaller average litter sizes as compared to females inoculated with saline. Females with unilateral tubal occlusion also tended to wean fewer pups than saline controls over the course of 90 days. Female mice with Chlamydia-induced tubal infertility mated more frequently (approximately every 11 d) than did fertile females (approximately every 20 d), which is consistent with the adaptive sterilization hypothesis. To determine whether Chlamydia-induced sterilization is truly adaptive, future studies will need to demonstrate increased sexual transmissibility, and possibly increased promiscuity, within populations of freely breeding mice.

Biological variation of serum thyrotropin and thyroid hormones concentrations determined at 8-week intervals for 1 year in clinically healthy cats.

BACKGROUND: Cats commonly develop thyroid disease but little is known about the long-term biological variability of serum thyroid hormone and thyrotropin (thyroid-stimulating hormone; TSH) concentrations. OBJECTIVES: We aimed to determine the long-term biological variation of thyroid hormones and TSH in clinically healthy cats. METHODS: A prospective, observational study was carried out. Serum samples for analysis of total thyroxine (T4, by radioimmunoassay [RIA] and homogenous enzyme immunoassay [EIA]), triiodothyronine (T3 ), free T4 (by dialysis), and TSH were obtained every 8 weeks for 1 year from 15 healthy cats, then frozen until single-batch analysis. Coefficients of variation (CV) within individual cats ( CV I ) and among individual cats ( CV G ), as well as the variation between duplicates (ie, analytical variation [ CV A ]) were determined with restricted maximum likelihood estimation. The indices of individuality (IoI) and reference change values (RCVs) for each hormone were calculated. RESULTS: Some thyroid hormones showed similar (total T4 by EIA) or greater (TSH) interindividual relative to intraindividual variation resulting in intermediate to high IoI, consistent with previous studies evaluating the biological variation of these hormones weekly for 5-6 weeks. By contrast, total T4 (by RIA) and free T4 had a low IoI. Total T3 had a high ratio of CV A to CV I ; therefore, interindividual variation could not be distinguished from analytical variation. No seasonal variability in the hormones could be demonstrated. CONCLUSIONS: Clinicians might improve the diagnosis of feline thyroid disease by establishing baseline concentrations for analytes with intermediate-high IoI (total T4, TSH) for individual cats and applying RCVs to subsequent measurements.

Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Sex Differences in Spotted Hyenas.

The apparent virilization of the female spotted hyena raises questions about sex differences in behavior and morphology. We review these sex differences to find a mosaic of dimorphic traits, some of which conform to mammalian norms. These include space-use, dispersal behavior, sexual behavior, and parental behavior. By contrast, sex differences are reversed from mammalian norms in the hyena's aggressive behavior, social dominance, and territory defense. Androgen exposure early in development appears to enhance aggressiveness in female hyenas. Weapons, hunting behavior, and neonatal body mass do not differ between males and females, but females are slightly larger than males as adults. Sex differences in the hyena's nervous system are relatively subtle. Overall, it appears that the "masculinized" behavioral traits in female spotted hyenas are those, such as aggression, that are essential to ensuring consistent access to food; food critically limits female reproductive success in this species because female spotted hyenas have the highest energetic investment per litter of any mammalian carnivore. Evidently, natural selection has acted to modify traits related to food access, but has left intact those traits that are unrelated to acquiring food, such that they conform to patterns of sexual dimorphism in other mammals.

Germ cell nests in adult ovaries and an unusually large ovarian reserve in the naked mole-rat.

The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35-40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen's suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species' protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR's long reproductive lifespan remains to be determined.

Spotted hyaenas and the sexual spectrum: reproductive endocrinology and development.

The spotted hyaena (Crocuta crocuta) is a unique species, even amongst the Hyaenidae. Extreme clitoral development in female spotted hyaenas challenges aspects of the accepted framework of sexual differentiation and reproductive function. They lack a vulva and instead urinate, copulate and give birth through a single, long urogenital canal that traverses a clitoris superficially resembling a penis. Recent and historical evidence is reviewed to describe our changing understanding of the biology of this species. Expanding upon observations from hyaenas in nature, much has been learned from studies utilising the captive colony at the University of California, Berkeley. The steroid environment of pregnancy is shaped by placental androgen and oestrogen secretion and a late gestational increase in sex hormone binding globulin, the regulated expression and steroid-binding characteristics of which are unique within the Hyaenidae. While initial external genital development is largely free of androgenic influence, the increase in testosterone concentrations in late gestation influences foetal development. Specifically, anti-androgen (AA) treatment of pregnant females reduced the developmental influence of androgens on their foetuses, resulting in reduced androstenedione concentrations in young females and easier birth through a 'feminised' clitoris, but precluded intromission and mating by 'feminised' male offspring, and altered social interactions. Insight into the costs and benefits of androgen exposure on spotted hyaena reproductive development, endocrinology and behaviour emphasises the delicate balance that sustains reproductive success, forces a re-evaluation of how we define masculine vs feminine sexual characteristics, and motivates reflection about the representative value of model species.

Evaluation of combined assessments of serum anti-Müllerian hormone and progesterone concentrations for the diagnosis of ovarian remnant syndrome in dogs.

OBJECTIVE: To determine the usefulness of combined assessments of serum anti-Müllerian hormone (AMH) and progesterone (P4) concentrations for diagnosis of ovarian remnant syndrome (ORS) in dogs. DESIGN: Prospective case series. ANIMALS: 602 bitches that had previously undergone ovariohysterectomy or ovariectomy without hysterectomy and that were being evaluated for ORS. PROCEDURES: Serum AMH and P4 concentrations were measured in a single serum sample obtained from each of the 602 dogs; results were classified as positive, negative, or inconclusive. Following AMH testing, submitting veterinarians were offered reimbursement for shipping and histologic evaluation of any tissue removed during treatment of dogs for which serum samples had been assessed. RESULTS: Tissue samples were provided from 53 of 55 dogs that underwent surgical abdominal exploration. Of 48 dogs with histologically confirmed ORS, 25 were positive for both AMH and P4 and 23 were positive for AMH or P4 or test results were inconclusive. No dogs with histologically confirmed ORS were negative for both AMH and P4. Tissue samples that included no ovarian tissue were obtained from 5 dogs that were positive for both AMH and P4. In 2 dogs that were negative for both AMH and P4, no ovarian remnant was identified and no tissue was removed at the time of exploratory surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that combined determination of AMH and P4 concentrations in a single serum sample can be an effective diagnostic test for spayed dogs suspected to have ORS. Histologic analysis of excised tissues to assess the completeness of surgical treatment in dogs with ORS is warranted.

Age-associated and deslorelin-induced declines in serum anti-Müllerian hormone concentrations in female cheetahs, Acinonyx jubatus.

Anti-Müllerian hormone (AMH) is widely used in human medicine to non-invasively estimate the size of the ovarian follicle reserve and to predict the ovarian response to gonadotropin stimulation in the context of assisted reproductive technologies (e.g., IVF). These applications of AMH testing have recently expanded to non-human mammals, with production animals, such as cows, goats and sheep being the primary focus of AMH research. However, few investigations have involved exotic species, and in particular carnivores. In this study, we measured AMH concentrations (0.078-3.078ng/mL) in archived serum samples that had been collected from 36 adult female cheetahs across their reproductive lifespan (2-15years of age). Similar to other mammals, AMH concentration in cheetahs declined with age, and its variability among females of the same age was considerable. The rates at which AMH declined over time in individual cheetahs were also highly variable. Five cheetahs had been contracepted with the long-acting GnRH agonist deslorelin for 6-18months prior to sample collection, and their AMH concentrations were relatively low compared to untreated females. In this first study of AMH in an exotic carnivore, the findings demonstrate that the age-associated decline in AMH is highly variable and that deslorelin appears to suppress AMH concentration in serum. Owing to the increased use of assisted reproductive technologies in ex situ populations of threatened and endangered species, such as cheetahs, the present study's findings will need to be taken into consideration if AMH is to be used successfully to optimize breeding management decisions in exotic species.

Leptin levels, seasonality and thermal acclimation in the Microbiotherid marsupial Dromiciops gliroides: Does photoperiod play a role?

Mammals of the Neotropics are characterized by a marked annual cycle of activity, which is accompanied by several physiological changes at the levels of the whole organism, organs and tissues. The physiological characterization of these cycles is important, as it gives insight on the mechanisms by which animals adjust adaptively to seasonality. Here we studied the seasonal changes in blood biochemical parameters in the relict South American marsupial Dromiciops gliroides ("monito del monte" or "little mountain monkey"), under semi-natural conditions. We manipulated thermal conditions in order to characterize the effects of temperature and season on a battery of biochemical parameters, body mass and adiposity. Our results indicate that monitos experience an annual cycle in body mass and adiposity (measured as leptin levels), reaching a maximum in winter and a minimum in summer. Blood biochemistry confirms that the nutritional condition of animals is reduced in summer instead of winter (as generally reported). This was coincident with a reduction of several biochemical parameters in summer, such as betahydroxybutyrate, cholesterol, total protein concentration and globulins. Monitos seem to initiate winter preparation during autumn and reach maximum body reserves in winter. Hibernation lasts until spring, at which time they use fat reserves and become reproductively active. Sexual maturation during summer would be the strongest energetic bottleneck, which explains the reductions in body mass and other parameters in this season. Overall, this study suggests that monitos anticipate the cold season by a complex interaction of photoperiodic and thermal cues.

Mate preference for dominant vs. subordinate males in young female Syrian hamsters (Mesocricetus auratus) following chemically-accelerated ovarian follicle depletion.

Life history theory predicts that selectivity for mates generally declines as females age. We previously demonstrated this phenomenon in Syrian hamsters (Mesocricetus auratus), in that older females showed reduced preference for dominant over subordinate males. To test the hypothesis that decreased reproductive quality due to aging reduces mate preference, we decoupled reproductive and chronological age by treating young female hamsters with 4-vinylcyclohexene diepoxide (VCD), which destroys ovarian follicles and functionally accelerates ovarian follicle depletion without compromising the general health of rodents. In this study, VCD effectively reduced follicle numbers in young Syrian hamsters. VCD-treated and control females were allowed to choose between a dominant and a subordinate male in a Y-maze on the day of proestrus. Both VCD-treated and control females demonstrated preference for the dominant male by leaving a greater proportion of vaginal scent marks near him, which is a behavior that females display when soliciting prospective mates. However, there was no effect of treatment on the proportion of vaginal scent marks left for the dominant male. Furthermore, ovarian follicle numbers were not significantly correlated with any behaviors in either group. We conclude that accelerated ovarian follicle depletion does not reduce mate preference in young female hamsters.

An assessment of anti-Müllerian hormone in predicting mating outcomes in female hamsters that have undergone natural and chemically-accelerated reproductive aging.

In mammals, female fertility declines with age due in part to a progressive loss of ovarian follicles. The rate of follicle decline varies among individuals making it difficult to predict the age of onset of reproductive senescence. Serum anti-Müllerian hormone (AMH) concentrations correlate with the numbers of ovarian follicles, and therefore, AMH could be a useful predictor of female fertility. In women and some production animals, AMH is used to identify which individuals will respond best to ovarian stimulation for assisted reproductive technologies. However, few studies have evaluated AMH's predictive value in unassisted reproduction, and they have yielded conflicting results. To assess the predictive value of AMH in the context of reproductive aging, we prospectively measured serum AMH in 9-month-old Siberian hamsters shortly before breeding them. Female Siberian hamsters experience substantial declines in fertility and fecundity by 9months of age. We also measured serum AMH in 5-month-old females treated with 4-vinylcyclohexene diepoxide (VCD), which selectively destroys ovarian follicles and functionally accelerates ovarian aging. Vehicle-treated 5-month-old females served as controls. AMH concentrations were significantly reduced in VCD-treated females yet many females with low AMH reproduced successfully. On average, both young and old hamsters that littered had higher AMH concentrations than females that did not. However, some females with relatively high AMH concentrations failed to litter, whereas several with low AMH succeeded. Our results suggest that mean AMH concentration can predict mating outcomes on a population or group level, but on an individual basis, a single AMH determination is less informative.

Socioecological variables predict telomere length in wild spotted hyenas.

Telomeres are regarded as important biomarkers of ageing and serve as useful tools in revealing how stress acts at the cellular level. However, the effects of social and ecological factors on telomere length remain poorly understood, particularly in free-ranging mammals. Here, we investigated the influences of within-group dominance rank and group membership on telomere length in wild adult spotted hyenas (Crocuta crocuta). We found large effects of both factors; high-ranking hyenas exhibited significantly greater mean telomere length than did subordinate animals, and group membership significantly predicted mean telomere length within high-ranking females. We further inquired whether prey availability mediates the observed effect of group membership on telomere length, but this hypothesis was not supported. Interestingly, adult telomere length was not predicted by age. Our work shows for the first time, to the best of our knowledge, the effects of social rank on telomere length in a wild mammal and enhances our understanding of how social and ecological variables may contribute to organismal senescence.

4-Vinylcyclohexene diepoxide reduces fertility in female Siberian hamsters when treated during their reproductively active and quiescent states.

The industrial compound 4-vinylcyclohexene diepoxide (VCD) destroys ovarian follicles and reduces fertility in rodents, but to date VCD has not been tested in species that experience seasonal anestrus. To determine if VCD destroys follicles when administered during reproductive quiescence, Siberian hamsters were treated with VCD (240mg/kg i.p. daily for 10 days) during short days, and outcomes were compared with reproductively active females that were maintained and treated in long days. Primordial follicle numbers were significantly reduced by VCD under both day lengths, and reproductive quiescence in short days did not appear to render the ovaries less susceptible to VCD-induced follicle depletion. Independent of day length and reproductive state, VCD-treated hamsters weaned substantially fewer offspring than controls. These results suggest that time of year may not be an important consideration for optimizing use of VCD in the field when the target pest species is a seasonally breeding rodent.

Dorsomedial hypothalamic lesions counteract decreases in locomotor activity in male Syrian hamsters transferred from long to short day lengths.

The dorsomedial nucleus (DMN) of the hypothalamus has been implicated in seasonal control of reproduction. Syrian hamsters with DMN lesions, unlike control hamsters, do not undergo testicular regression after transfer from a long day length (14 h of light per day; LD) to a short day length (8 h of light per day; SD). SDs also markedly reduce hamster locomotor activity (LMA). To assess whether the DMN is a component of the neural circuitry that mediates seasonal variation in LMA, neurologically intact males (controls) and hamsters that had sustained lesions of the DMN (DMNx) were housed in an LD or SD photoperiod for 26 weeks. DMNx that prevented testicular regression counteracted decreases in LMA during 8 to10 weeks of SD treatment; steroid-independent effects of SDs did not override high levels of LMA in DMNx males. As in previous studies, testosterone (T) restoration increased LMA in LD but not SD castrated control males. In the present study, T also failed to increase LMA in SD-DMNx hamsters. The DMN is not necessary to maintain decreased responsiveness of locomotor activity systems to T in SDs, which presumably is mediated by other central nervous system androgen target tissues. Finally, DMNx did not interfere with the spontaneous increase in LMA exhibited by photorefractory hamsters after 26 weeks of SD treatment. We propose that DMN is an essential part of the substrate that mediates seasonal decreases in LMA as day length decreases but is not required to sustain decreased SD responsiveness to T or for development of refractoriness to SDs.

Reduced mate preference for dominant over subordinate males in old female Syrian hamsters (Mesocricetus auratus).

Why some females choose to mate with a 'preferred' male, whereas others choose to mate with an 'inferior' male is not always clear. Generally, the choosiness of females is thought to decline with advanced age, but relatively few studies have investigated this concept, and reports of this phenomenon in mammals are lacking. To address this deficiency, young and old female golden hamsters were evaluated for their preference for dominant vs. subordinate males. Females observed male dyads as a dominance relationship was established. Dominant and subordinate males were then placed within enclosures at the opposite ends of a Y-maze, and the first approach, scent marking, and time spent near each male were evaluated in young and old females during pro-oestrus-a time when females solicit visits by prospective mates by leaving vaginal and flank scent marks. Whereas the proportion of time spent near the dominant male was significantly greater than random for both young and old females, the proportions of vaginal and flank scent marks left for the dominant male were significantly greater than random for young females, but not for old females. Overall, these results are consistent with a decline in the preference for dominant males by old female hamsters.

The effects of poly-unsaturated fatty acids on the physiology of hibernation in a South American marsupial, Dromiciops gliroides.

Many mammals hibernate, which is a profound lethargic state of several weeks or months during winter, that represents a transitory episode of hetherothermy. As with other cases of dormancy, the main benefit of hibernation seems to be energy saving. However, the depth and duration of torpor can be experimentally modified by the composition of food, especially by fattyacid composition. In eutherians, diets rich in unsaturated fatty acids (i.e., fatty acids with at least one double bond) lengthen torpor, reduce metabolism and permit hibernation at lower temperatures. Here we studied whether diets varying in fatty acid composition have an effect on the physiology of hibernation in a South American marsupial, Dromiciops gliroides. We designed a factorial experiment where thermal acclimation (two levels: natural versus constant temperature) was combined with diet acclimation: saturated (i.e., diets with high concentration of saturated fatty acids) versus unsaturated (i.e., diets with high concentration of unsaturated fatty acids). We measured energy metabolism in active and torpid individuals, as well as torpor duration, and a suite of 12 blood biochemical parameters. After a cafeteria test, we found that D. gliroides did not show any preference for a given diet. Also, we did not find effects of diet on body temperature during torpor, or its duration. However, saturated diets, combined with high temperatures provoked a disproportionate increase in fat utilization, leading to body mass reduction. Those animals were more active, and metabolized more fats than those fed with a high proportion of unsaturated fatty acids (="unsaturated diets"). These results contrast with previous studies, which showed a significant effect of fatty acid composition of diets on food preferences and torpor patterns in mammals.

Development of the external genitalia: perspectives from the spotted hyena (Crocuta crocuta).

This review/research paper summarizes data on development of the external genitalia of the spotted hyena, a fascinating mammal noted for extreme masculinization of the female external genitalia. The female spotted hyena is the only extant mammal that mates and gives birth through a pendulous penis-like clitoris. Our studies indicate that early formation of the phallus in both males and females is independent of androgens; indeed the phallus forms before the fetal testes or ovaries are capable of synthesizing androgens. Likewise, pre- and postnatal growth in length of the penis and clitoris is minimally affected by "androgen status". Nonetheless, several internal morphologies, as well as external surface features of the phallus, are androgen-dependent and thus account for dimorphism between the penis and clitoris. Finally, estrogens play a critical role in penile and clitoral development, specifying the position of the urethral orifice, determining elasticity of the urethral meatus, and facilitating epithelial-epithelial fusion events required for proper formation of the distal urethra/urogenital sinus and prepuce. Accordingly, prenatal inhibition of estrogen synthesis via administration of letrozole (an aromatase inhibitor) leads to malformations of the glans as well as the prepuce (hypospadias). The effects of prenatal androgens, anti-androgens and impaired estrogen synthesis correlated with the tissue expression of androgen and estrogen receptors.