Functional infertility among territorial males in two passerine species, the willow warbler Phylloscopus trochilus and the bluethroat Luscinia svecica

Information about male infertility in free-living birds is scarce, but anecdotal and circumstantial evidence suggests that it does occur regularly at a low frequency. In this paper we document three cases of azoospermia in two passerine species, the willow warbler Phylloscopus trochilus and the bluethroat Luscinia svecica at their breeding grounds in South Norway. In willow warblers, two males out of a sample of 50 territory holders had no sperm in their seminal glomera, the storage site of sperm ready for ejaculation. The two males also had very small testes. One out of 48 bluethroat males also had no sperm in the seminal glomera. This male had an extreme asymmetry of the testes, with the right testis being about twice as large as the left. He also failed to fertilize any eggs in his own nest, as well as in neighbouring nests, as revealed by microsatellite genotyping. Thus, the proportion of males without sperm seems to be at a magnitude of a few (2–4) percent in both species. These are among the first estimates of the frequency of azoospermia in wild birds. Our results indicate a significant risk for sexually monogamous females of laying unfertilized eggs, which could favour the evolution of extra-pair copulation as a fertility insurance strategy in females.     

Copulation induces an acute increase in epididymal sperm numbers in rats

The temporal changes in epididymal sperm numbers during the month after sexual contact were examined in 80 adult albino rats. The comparably sexually experienced males in 6 experimental groups were allowed 3 ejaculations with ovariectomized females in which oestrus was induced by hormone injections. Epididymides were removed from the experimental males 1 h, 1 day, 2 days, 3 days, 7 days or 30 days later. Control males were sexually inexperienced or sexually experienced and living with females. Epididymal sperm counts revealed a pattern that resembled an inverted V-shaped function with a peak at 48 h after copulation. We suggest that sexual activity provokes an acute attenuation of sperm disposal within the epididymis with the result that more spermatozoa are available for ejaculation during sexually active times.     

The Effect of Aluminum Exposure on Reproductive Ability in the Bank Vole (<Emphasis Type="Italic">Myodes glareolus</Emphasis>)

Human impact on the environment is steadily increasing the amounts of aluminum in the ecosystems. This element accumulates in plants and water, potentially exposing herbivores to its harmful effect. In heavily polluted sites, a decrease in the density of small rodent populations has been observed. This decline may be caused by many factors, including decreased fertility. The aim of the presented research was to determine how aluminum, administered at concentrations similar to those recorded in industrial districts (Al I = 3 mg/l, Al II = 200 mg/l), affects the reproductive abilities of small rodents. As the indicators of reproductive abilities, body weight, weight of the testes and accessory sex glands of males, and uterus weight of females were estimated. In females, the number of matured follicles (types 6, 7, and 8) was analyzed, while in males, the quantity and quality (matured, viable, swollen, motile, head abnormalities) of epididymal sperm cells were assessed. Moreover, the development of testes, measured by spermatogenic index, was determined. The model species was the bank vole. Our results have proven that aluminum impairs adult individuals’ reproductive abilities by decreasing the quality and quantity of sperm cells and by causing morphologically abnormal development of the gonads. However, no difference in male organometric parameters was found, and only in females treated with 3 mg/l Al, the uterus weight was higher than control. No differences were found in the total number of matured follicles. These results suggest that the decline in rodent numbers in industrial districts is due, at least in part, to poorer males’ reproductive abilities, resulting from exposure to aluminum contamination.     

Effect of antibodies to human seminal plasma inhibin on spermatogenesis and sperm agglutination in adult male rats

In vitro incubation of rat epididymal sperm with antiserum to human seminal plasma inhibin (As hSPI) caused agglutination of the sperm. In vivo administration of As hSPI to adult male rats resulted in a significant decrease in testicular as well as epididymal sperm counts. Furthermore, the majority (almost 90%) of the epididymal sperm were agglutinated. When these animals were mated with normal cycling females, significant reduction in fertility was observed.     

Seasonal reproduction of yellowish myotis,Myotis levis (chiroptera: Vespertilionidae), from a Neotropical highland

With a nearly global distribution the vespertilionid bat Myotis represents one of the most exceptional examples of adaptive radiation among mammals. We investigated the reproductive activity of the vespertilionid bat yellowish myotis, Myotis levis, from a highland area in Southeastern Brazil. The data were obtained through histological analyses of the male and female genital systems from February 2010 to May 2011. The testes of the adult yellowish myotis showed seasonal morphological characteristics which were categorized in the following stages: rest, maturing, mature, and mating. Rest and maturing males were recorded throughout the rainy season (October‐March). In the rest stage no spermatogenesis was observed and the epididymal duct was devoid of spermatozoa. Maturing individuals had started spermatogenesis and few spermatozoa were found in the epididymal duct. Mature males were found toward the end (February‐March) of the rainy season, when full spermatogenic activity was recorded and spermatozoa were packed in the epididymal duct. Although not recorded, mating probably occurred in the middle of the dry season (April–September) when the cauda epididymis was enlarged and packed with sperm. The spermatozoa remained stored in the cauda epididymis for at least three months when the testes entered into regression. The ovaries showed all types of ovarian follicles throughout the study period except mature follicles which were registered only in July (mid‐dry season). Lactating females were captured in the beginning of the rainy season. The seasonal reproductive characteristics of the yellowish myotis from this Neotropical highland area were similar those of epididymal sperm‐storing temperate vespertilionids. J. Morphol. 274:1230–1238, 2013. © 2013 Wiley Periodicals, Inc.     

Factors affecting female sexual receptivity in the planthopper, Prokelisia dolus

Abstract. The mating system of Prokelisia dolus Wilson (Homoptera: Delphacidae) was characterized by determining: if males and females multiply mate; when transitions occur in female sexual receptivity, what triggers sexual refractoriness; and what behaviours characterize unreceptive virgins, receptive virgins, and unreceptive mated females. Males copulated with up to six females in less than 1 h, but completely inseminate, on average, only the first four females. Females rarely mated more than once, unless males were depleted of sperm due to previous copulations or if copulation was interrupted (if duration was<2 min). Male and female calling was associated (100% and 91%, respectively) with sexual receptivity and resultant matings. The transition from unreceptive virgin to receptive (calling) mature virgin occurred 48 h posteclosion, and all were mated by day 4. Females that were sexually immature and those completely inseminated did not call. Rejection of males by females included walking away from approaching males (65%), female kicking (7%), and abdominal lifting (5%). Rejection of males was observed by immature, mature and calling, and mated females. Sexual refractoriness was not triggered by acoustic and visual stimuli or mechanical stimulation of genitalia. Refractoriness was also not triggered by reception of small quantities of sperm because some females laid a few viable eggs yet calling was not terminated. Sexual refractoriness was activated by a substance in the ejaculate as demonstrated by injection into the haemocoel of male accessory glands or testes and homogenates of seminal vesicles. This is the first study that documents the role of male ejaculate in inhibiting female sexual receptivity in Hemiptera (Homoptera).     

Mating behaviour and insemination in the hopping mouse (Notomys alexis)

Hopping mice have extremely small testes and a reduced complement of male accessory sex glands. Nevertheless, greater than 1000 spermatozoa populate the isthmus of the oviduct after each insemination and a range of morphological types is found similar to that in the male tract. When females are primed with gonadotrophins they sometimes lock with more than one of the males within the group and in the present study two inseminations sometimes took place at the one oestrus. There is thus the possibility for intermale sperm competition within the female tract of these animals.     

Xenografting of adult mammalian testis tissue

Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences.     

Effects of expression of human or bovine growth hormone genes on sperm production and male reproductive performance in four lines of transgenic mice.

Reproductive performance was studied in transgenic males from lines expressing and transmitting four hybrid genes: mouse metallothionein-I/human growth hormone (GH) (MT/hGH), MT/hGH placental variant (MT/hGH.V), MT/bovine GH (MT/bGH) and phosphoenolpyruvate carboxykinase/bGH (PEPCK/bGH). Each male was exposed to three normal females for 1 week and to three different normal females for another week. Females were examined for vaginal plugs and necropsied on day 14 of pregnancy. Males were killed for analysis of organ weights, numbers of testicular spermatids, numbers of epididymal sperm and measurements of plasma glucose concentration. Fertility of MT/hGH and MT/hGH.V transgenic males was significantly lower than in normal males, primarily because most males failed to impregnate any females. In females that became pregnant, the numbers of corpora lutea, total fetuses and live fetuses did not differ from those in females mated to normal (nontransgenic) males. Fetal crown-rump length on day 14 of pregnancy did not differ between litters sired by normal or by transgenic males. Weights of testes and seminal vesicles were significantly greater in all four types of transgenic male, but daily sperm production per unit weight (g-1) of testis was not affected and epididymal sperm reserves were either normal or slightly higher than normal. Plasma glucose concentrations were significantly higher in PEPCK/bGH mice than in other mice. Average or individual reproductive performance of transgenic males from the various lines did not correlate with any of the parameters examined except for significantly heavier seminal vesicles in MT/hGH and MT/hGH.V males than in normal males; these transgenic males exhibited a high incidence of infertility. Since hGH and hGH.V, but not bGH, are lactogenic in rodents, it was concluded that chronic stimulation of GH and prolactin receptors by ectopically produced human GHs in transgenic mice compromises male fertility by an unknown mechanism. Reduced fertility of transgenic males with MT/hGH or MT/hGH.V hybrid genes is due to failure to inseminate or impregnate females rather than to reduced numbers of spermatozoa or gross changes in the male reproductive system.     

The influence of sex,sexual condition and age on the exploratory behaviour of wild wood mice (Apodemus Sylvaticus L.)

From April 1978 to April 1980 the exploratory behaviour of more than 1000 wild wood mice was studied in a modified open field test situation. The influence of sex, sexual condition and age on the exploratory activity was investigated. Sufficient data being available, the exploratory activity of both sexually active and sexually inactive adult males and adult females, of both sexually active and sexually inactive subadult males and subadult females and of both sexually inactive juvenile males and females was compared by means of a one way analysis of variance for each two-month period. In other analyses the influence of age of adult males and adult females on the exploratory behaviour was further examined. No differences could be found in the exploratory activity displayed by males or females and by sexually active or sexually inactive animals. Also a clear influence of age was absent. The interindividual variation was very large.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

Absence of female-induced luteinizing hormone release in orchidectomized, sexually active mice.

This experiment was designed to determine whether the reflexive LH pulses induced in male mice by exposure to a female are dependent upon the presence of the testes. B6D2F1 males were studied because, unlike most males, these hybrids remain sexually active indefinitely after castration, demonstrating that they still recognize females and are sexually arousable. We reasoned that if LH reflexes occur independently of the testes, then exposing castrated B6D2F1 males to a female should elicit a pulse. We observed female-induced pulses in 67% of intact, naive males and 80% of intact, sexually experienced males, but they were absent in castrated, sexually experienced males. Spontaneous pulses were too frequent in castrated, naive males to distinguish potential LH reflexes. Thus, the induction of LH reflexes depends upon the presence of the testes in these hybrid males. Secondarily we found more frequent spontaneous pulses in sexually naive than in experienced mice regardless of their gonadal status. These unexpected results imply that long-term mating activity slows the pace of spontaneous LH pulses in these hybrids. Considered together, the differential effects of the testes and sexual experience on both types of LH pulses suggest that two distinct pulse-generating mechanisms exist in male mice.     

Effect of nutrition, temperature and photoperiod on the rate of sexual maturation of the field vole (Microtus agrestis)

Weanling male and female field voles were placed in long or short photoperiods, kept at 18 degrees C or 4 degrees C, and fed (ad libitum) diets containing 24%, 16%, 8% and 4% protein, for 6 weeks. Animals in the long photoperiod were more sexually mature than were animals in the short photoperiod. Temperatures had no effect on females, but did affect males: those kept at 4 degrees C had heavier testes and wider seminiferous tubules than those kept at 18 degrees C. There was little difference between the animals on 24%, 16% and 8% protein diets. Animals on 4% protein diets had retarded growth rates and were significantly less sexually mature than those on the other 3 diets, males having smaller testes and seminal vesicles and narrower seminiferous tubules and females having smaller ovaries and uteri.     

Murine cytomegalovirus infection of mouse testes.

With the aim of illustrating a mechanism of cytomegalovirus (CMV) venereal transmission, we induced murine CMV infection in the mouse testes of immunologically competent mice. Using in situ cytohybridization, we were able to show that murine CMV-specific DNA was associated with spermatocytes and mature sperm. Electron microscopy studies also supported sperm infection. The virus could be reisolated from infected epididymal sperm by cocultivation with mouse embryo fibroblasts. We found no difference in either the sexual performance or the fertilization efficiency of the sperm between infected and uninfected males.     

Are secondary sex traits, parasites and immunity related to variation in primary sex traits in the Arctic charr?

Primary and secondary sex traits are influenced by the same sex hormones, and the expression of secondary sex traits may consequently signal males' capacity for sperm production. Sperm quality may also be influenced by immune activity, as sperm are non-self to the male. Parasite infections alter immune activity and may thus reduce ejaculate quality. In the Arctic charr (Salvelinus alpinus) the red abdominal colour is considered an ornament that signals important information in mate choice. We captured and individually caged sexually mature male Arctic charr during the spawning period. Afterwards we estimated abdominal colour, parasite infections, gonad mass and several spermatological and immunological variables. Intensity of abdominal colour was positively correlated to testes mass, milt mass and sperm cell numbers produced. Additionally, males with low parasite intensities had high testes mass and produced milt with high sperm density, indicating a trade-off between parasite resistance and development of primary sex traits. Our measures of immunity were, however, not related to primary sex traits. We conclude that females evaluating male abdominal coloration may obtain information about differences between males in fertilization potential and parasite resistance.     

Male Mating History Influences Female Mate Choice in the Trinidadian Guppy (Poecilia reticulata)

Based on the phenotype‐linked fertility hypothesis, sexual selection should favour females that can accurately assess the recent mating history of available sexual partners and preferentially avoid mating with recently‐mated males [who may be sperm depleted (SD)] so as to minimize the risk of their eggs not being fertilized. This hypothesis has received to date only limited attention and empirical support. Therefore, in the current study, we investigated experimentally whether females of a vertebrate species, the Trinidadian guppy (Poecilia reticulata), are able to assess the recent mating history of males, and thus potentially their functional fertility, and choose to avoid mating with males that appear to have recently mated and who may be sperm limited. Individual virgin females were first given a dichotomous choice between a male that had not been recently observed to interact sexually with another female (i.e. not sperm‐depleted) and another male that had been observed to interact sexually with a female (i.e. potentially sperm‐depleted) as sexual partners. Paired males were matched for body length and coloration. Immediately following this test, the focal females were subjected to a free‐swimming mate‐choice test using the same paired stimulus males. As predicted, on average, female guppies avoided the apparently recently‐mated (and potentially sperm‐depleted) male and exhibited a significant preference for the other male not recently observed mating (and thus not likely sperm limited) during both tests. We do not yet fully understand the underlying mechanisms of this preference. Therefore, further research on the particular cues that females use to assess the recent mating history and fertility status of males is required.     

Development of the normal XY male and sex-reversed XXSxr pseudomale mouse epididymis

XXSxr pseudomale mice (chromosomally XX animals "sex-reversed" by the Sxr factor) develop testes and produce sufficient androgens for masculinization as assessed at the macroscopic level. However, adult XXSxr pseudomales lack the epididymal initial segment (I.S.). In this study prenatal and postnatal epididymal development was examined histologically and biochemically, and it was found that XXSxr pseudomales are indistinguishable from normal XY males up to day 21 of postnatal life. By 25 days postnatally, before the onset of the pubertal androgen surge, the I.S. precursor is evident in normal animals but absent in XXSxr mutants. No major abnormalities were seen in other segments of the XXSxr epididymis. Our data suggest that androgen levels in testis and epididymis are not higher in normal XY males than in XXSxr pseudomale mice of the same age. Inadequate availability of androgens at the target site is unlikely to be the cause of the epididymal abnormality in XXSxr pseudomale mice.     

Epididymis as a target for contraception

Advantage of using a vaccine based on sperm antigens is that it can be used both in males and females as individuals who have antisperm antibodies are usually infertile but otherwise healthy. Several sperm specific antigens identified as prospective candidates for immunocontraception are of testicular origin. For the purpose of immunocontraception it may be desirable not to disrupt spermatogenesis and testicular function. Concept of post testicular maturation of spermatozoa has been very well established. During post testicular voyage spermatozoa undergo a series of complex and sequential events which transforms the immature immotile spermatozoa into mature sperm. Acquisition of functional maturity is necessary for progressive motility, zona pellucida recognition culminating in sperm egg binding. Importance of epididymal maturation is highlighted by the fact that high percentage of male infertility in human originates from the malfunction of the epididymis. The epididymis has also shown to be involved in sperm storage and provides an adequate environment for final maturation of the sperm. It provides a conducive microenvironment by virtue of which the spermatozoa are protected during the storage. In view of this it is imperative that more attention needs to be focused on epididymal antigens. The information obtained will enable us to identify epididymal antigens relevant to fertility and also help in infertility diagnosis.     

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.     

Sperm competition and male ejaculate investment in Nauphoeta cinerea: effects of social environment during development

Selective pressure arising from sperm competition has been predicted to influence evolutionary and behavioural adjustment of ejaculate investment, but also may influence developmental adjustment of ejaculate investment. Immature males able to target resources strategically based on the competitive environment they will experience when they become sexually mature should be at a selective advantage. In our study we investigated how the presence of potential competitors or mates affects ejaculate and testes investment during development in the cockroach Nauphoeta cinerea, a species where males control female remating via their ejaculate size (large spermatophores prevent females from remating and therefore function to avoid sperm competition for males) and females store sperm. Our aim was to determine whether the social environment influences developmental adjustment of ejaculate investment and the relative importance of ejaculate components with different functions; avoidance of or engagement in sperm competition. We conclude that the social environment can influence developmental and behavioural flexibility in specific ejaculate components that may function to avoid or engage in sperm competition.