Hormonal synchronization of the menstrual cycles of pigtail macaques to facilitate biomedical research including modeling HIV susceptibility

Background Menstrual cycle synchronization of female pigtail macaques could prove an invaluable resource in studies of the reproductive tract, associated infections, and other potential research fields. We tested whether use of an oral progesterone and estradiol combination tablet could synchronize menstrual cycles following treatment discontinuation. Methods Daily desogestrel 0.075 mg and ethinyl estradiol 0.01 mg were administered orally to three pigtail macaques at visual onset of perineal sex swelling and were continued until all animals had received it for at least 45 days. The hormones were discontinued, and these three macaques and three controls were observed for menstruation and had blood progesterone and estrogen measured over an additional 2‐month period. Results All treatment animals showed spontaneous menstrual cycle synchronization for 2 months after menstrual cycling resumed. Conclusion Progesterone and estradiol combination therapy can be used in pigtail macaques to induce synchronized cycling that persists in the absence of on‐going hormone treatments.     

Potential applications of electrical impedance techniques in female mammalian reproduction

The measurement of electrical impedance is one of many methods that can be used for monitoring various reproductive events in female mammals. This paper summarizes the key findings in this research area that have been achieved during the past four decades. The electrical impedance in the vagina, vaginal vestibule and vulva during the estrous/menstrual cycle shows significant changes that vary among these reproductive organs, different locations in these organs and mammalian species. The changes of vaginal and vulvar impedance during periestrus are temporarily associated with the preovulatory luteinizing hormone (LH) peak and are significantly correlated with systemic levels of estradiol and progesterone. In humans, significant impedance changes during the menstrual cycle and their close association with the LH peak and ovulation have been found not only in the vagina, but also in the tongue. Findings of a number of studies suggest the possibility of using vaginal, vestibular and vulvar impedance to predict and confirm the fertile period of the estrous/menstrual cycle. There is some evidence that vulvar and cervical impedance may be a reliable indicator of impending parturition. Results in several studies also indicate the possibility of using electrical impedance methods to confirm the existence of ovarian follicular cysts, endometriosis and cervical neoplasia. However, physical and biological factors that may affect the impedance variation in the female reproductive system and tongue are still poorly understood. Their detection allows us to improve our ability to predict more precisely various reproductive processes by impedance techniques. This review also provides some considerations about the origin of impedance changes in the female reproductive system and tongue that may be useful for the systematic development of impedance methods in the field of female mammalian reproduction.     

动物生殖抑制研究进展：理论模型、方式和机制

生殖抑制指原本具有生育能力的动物个体因特定外界环境或生理条件而减少或丧失生殖能力的现象,有时是受环境变化的主动调控,更多的是出现在其他个体影响下的被动抑制,极端情况发生在社会性动物的永久性抑制,即永久无法生殖或无法生殖成熟。研究发现非社会性动物也有生殖的推迟及可恢复性的生殖抑制,生殖抑制影响着动物种群数量动态、维持和进化。随着多学科的发展,生殖抑制机理研究取得了诸多进展。从阐述生殖抑制的概念出发,解析生殖抑制的形态、激素和分子生理特征,总结了生殖抑制的原因、作用,终述现有的理论模型以及不同物种方面的最新进展,并就生殖抑制在生物资源管理方面的应用价值进行了展望,旨在丰富生殖抑制的理论,扩展应用实践,为后续的生物资源管理提供理论参考。     

Birth of viable puppies derived from breeding cloned female dogs with a cloned male

Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo–matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.     

First postpartum estrus and pregnancy in the female collared peccary (Tayassu tajacu) from the amazon

The onset of sexual cycle postpartum was described in the collared peccary (Tayassu tajacu). Serum progesterone and 17beta-estradiol profiles, vaginal smears and external genitalia were analyzed in 20 animals housed with their piglets during the first postpartum month. The appearance of external genitalia showed no variation in any of the females: a shallow, reddish vulva, and vaginal mucus were constant features throughout the study. Based on hormonal profiles and vaginal smear cell patterns, 16 (80%) of the 20 peccaries showed signs of estrus and were considered cycling. The remaining four females (20%) did no show signs of estrus confirmed by low levels of progesterone (0.9+/-0.4 ng/mL) during the first postpartum month. In the cycling peccaries, a serum 17beta-estradiol peak (53.4+/-8.1 pg/mL) was observed on Day 7+/-1 postpartum, along with a linear increase in progesterone concentration from 3 (4.3+/-2.6 ng/mL) to 11 (30.8+/-4.9 ng/mL) days after this estradiol peak. Proportions of the different cells of the vaginal epithelium also changed in these females: superficial plus intermediate cells amounted to 76% of the cell total between Days 6 and 9 postpartum, corresponding to the estradiol peak. Nine (56%) of the 16 cycling females mated, indicated by the presence of sperm cells in their vaginal smears, and 6 (67%) became pregnant, reaching term. Non-pregnant cycling females (n=10) showed a steady decrease in serum progesterone concentration from 11 to 23 days after the estradiol peak, when basal levels were attained and a new estradiol peak registered, indicating the resumption of cyclicity in these females. The time interval between the two estradiol peaks was 23.5+/-2.1 days in these females. In pregnant females, progesterone concentrations continued to rise to levels of 60 ng/mL (n=6) 23 days after mating. These findings indicate that the lactating collared peccary female can become cycling and fertile during the early postpartum period, and that a predominance of superficial plus intermediate vaginal cells can be taken as the first sign of estrus.     

Specific IgA and IgG antibodies in the secretions of the female reproductive tract: effects of immunization and estradiol on expression of this response in vivo

Uterine and vaginal secretions collected from intact adult female rats were analyzed to determine whether immunization at sites distal to the reproductive tract had any effect on the presence of specific IgA and IgG antibodies in genital tract secretions. Peyer's patch and i.p. immunization and boost with sheep red blood cells (SRBC) stimulated the appearance of specific IgA antibodies in uterine and vaginal secretions of uterine-ligated animals. IgG antibodies were also induced in uterine but not in vaginal secretions. In contrast, subcutaneous immunization and boost elicited a weak IgA uterine and IgG vaginal response. To establish the role of estradiol in regulating the presence of specific antibodies in the female genital tract, ovariectomized rats received primary and/or secondary Peyer's patch immunizations with hormone treatment. Administration of estradiol daily for 3 days before sacrifice resulted in a significant accumulation of IgA and IgG antibodies to SRBC in uterine secretions. In the absence of estradiol, antibody content was negligible. Vaginal antibody levels were also clearly influenced by estradiol. In contrast to the uterus, however, specific IgA and IgG antibodies were present in the vaginal secretions of saline-injected immunized animals and were markedly inhibited in animals treated with estradiol. These results indicate that antibodies in genital tract secretions can be induced by immunization of the Peyer's patches and that their presence in uterine secretions is clearly dependent on estradiol. Further, they indicate that gut-derived specific antibodies enter the vagina in the absence of hormone stimulation and that estradiol exerts an inhibitory effect on their presence in vaginal secretions.     

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.     

Performing Vaginal Lavage,Crystal Violet Staining,and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.     

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years^18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies^33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors³³.An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida^{3,4,11,16,17,19,21,25,37}. This model has also been employed to test potential antifungal therapies in vivo^13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection^6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties^3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.     

Reproductive experience alters anxiety-like behavior in the female rat

Reproductive experience (i.e. pregnancy and lactation) results in significant alterations in subsequent hormone levels in female rats. Several studies have demonstrated that circulating hormones can significantly affect anxiety-like behavior. Thus, the purpose of the present study was to determine whether reproductive experience induces alterations in anxiety-like behaviors in cycling female rats and in older, reproductively senescent rats. In Experiment 1, the elevated plus maze (EPM) was used to test young cycling (6-8 weeks post-weaning) and middle-aged (32-36 weeks post-weaning) primiparous rats and their age-matched nulliparous counterparts for anxiety-like responses. In Experiment 2, activity in the open field was used as an additional measure of anxiety-like behavior in young (proestrus) and middle-aged (constant estrus) primiparous and nulliparous rats. For Experiment 3, EPM testing was conducted in separate groups of young and middle-aged animals tested two weeks after ovariectomy. The results revealed that during proestrus, primiparous animals exhibited fewer anxiety-like behaviors on the EPM compared to nulliparous controls. In middle-aged animals, however, parity was associated with increased anxiety-like behavior. In the open field, young, non-lactating primiparous animals again exhibited fewer anxiety-like behaviors compared to nulliparous controls, an effect that was reversed in middle-aged animals. Effects of reproductive experience on the EPM in both age groups were eliminated by ovariectomy. Overall, the findings indicate that reproductive experience significantly alters anxiety-like behavior, effects that are influenced by the endocrine status and/or age of the female.     

Opiate antagonist counteracts reproductive inhibition by porcine ACTH extract.

The effects of naloxone, an opiate receptor antagonist, on the maturation of female mice chronically pretreated with ACTH from weaning, were examined. Naloxone treatment, begun at 40 days of age, blocked ACTH-induced delay of puberty. Naloxone also reversed decreases in weights of the reproductive organs produced by ACTH. Naloxone treatment also resulted in estrous cycling and increased the number of ACTH-treated mice with ovarian corpora lutea. These changes are reflected in increased LH levels in ACTH-naloxone treated vs. ACTH-saline treated animals. Naloxone alone may increase reproductive function. Effects of ACTH on spleen and thymus weights, presumably via the adrenals, were partially reversed by naloxone. ACTH may have a different mechanism in adrenal function as compared to reproductive function.     

Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse,rat, and hamster

Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.     

Isolation and identification of fungi from vaginal flora in three species of captive Leontopithecus

The ability to reproduce in captivity is an essential component of lion tamarin (Leontopithecus) conservation programs. However, infections such as vaginitis, cervicitis, and endometritis are important diseases that may influence the reproduction of these animals. Therefore, it is important to detect continuous or occasional vaginal microbial populations, and to understand their potential role as an endogenous source of infection [Collins, 1964; Blue, 1983; Pugh et al., 1986]. Vaginal swabs were collected from 25 female tamarins of the three currently available species (L. rosalia, L. chrysopygus, and L. chrysomelas) at the Center of Primatology in Rio de Janeiro, Brazil. The swabs were processed according to standard mycological protocols, and isolates were biochemically characterized. Fungal isolates were recovered from 16 animals (64.0%). The results showed that 70.6% of the isolated microorganisms consisted of yeast, including three species of Candida (mainly C. glabrata). We suggest that this species is a resident member of the normal vaginal flora in Leontopithecus. Filamentous fungi (mainly from Trichosporon, Aspergillus, and Penicilliumgenera) constituted 29.4% of the isolates, and were considered to be transitory contaminants of the genital area. We suggest that colonization of the vaginal environment is related to the endocrine pattern associated with the reproductive status of these animals, but not to parity.     

A benign method for maintaining ovulatory estrogen levels in cycling rhesus macaques

The goal of this study was to develop a relatively noninvasive technique for generating ovulatory estrogen levels in cycling females over extended periods of time. Eleven intact cycling rhesus macaques were given weekly injections of estradiol cypionate in an effort to obtain weekly levels which approximated ovulatory levels. A dose of 500 μg generated weekly estrogen values averaging 370 ± 18 pg/ml. This method is a satisfactory alternative to more traditional techniques. It involves no surgical procedures, it is relatively nonintrusive, it does not terminate a female's reproductive career, and it is not as time-consuming as daily injections. Female cycle state can be altered with a minimum of manipulation for studies involving the behavior of estrous females.     

Patterns of behavior across reproductive states of free-ranging female black-handed spider monkeys (Ateles geoffroyi)

Reports on the behavior of spider monkeys (genus Ateles) describe a suite of 5-6 behaviors that are indicative of an estrous female. This study presents hormonal data as an independent measure of reproductive state to determine if these behaviors are associated with any particular reproductive state or stage of the ovarian cycle. Fecal samples collected from 6 free-ranging female black-handed spider monkeys (Ateles geoffroyi) over the course of 11 months were assayed for estrogen (E1C) and progesterone (PdG) metabolites, using an enzyme immunoassay. Behavioral data collected from the same females were analyzed for patterns associated with different reproductive states. A more detailed analysis of behavioral data associated with reproductively cycling periods determined whether each behavior clustered with copulations or a particular phase in the ovarian cycle. Observations of place-sniffing were more frequent for 4 of 5 females when reproductively cycling. Of all copulations observed, 80% occurred when the female participant was reproductively cycling. In addition, the rates of self-clitoral hold and self-clitoral rub behaviors were significantly higher for 2 of 4 and 4 of 5 of females, respectively, when reproductively cycling. No behavior consistently occurred in association with copulations or with any particular phase in the ovarian cycle. Results from this study suggest that the behaviors tested in this paper can provide some information regarding female spider monkey reproductive state (whether or not she is reproductively cycling), but that they are not indicators of estrus in female spider monkeys.     

Environmental control of teleost reproductive cycles: a brief review

Reports concerned with the environmental regulation of teleost reproductive cycles are critically reviewed. Photoperiod and temperature have been the most frequently studied environmental factors with respect to control of sexual cycling. Photoperiod may exert the dominant regulatory role in the salmonid family, but few species have been examined so no conclusive statement is presently possible. Both photoperiod and temperature may be important controlling factors in the cyprinid family, but inadequate controls prohibit meaningful interpretation. Temperature may be extremely important in regulating reproductive cycles in the cyprinodontiform fishes. Although the data are inconclusive, it appears that changes in daylength play some role in reproductive cycling in the gasterosteids. Photoperiod and temperature seem to interact in regulating the sexual cycles in the 2 periform species investigated.
It is concluded that short term laboratory studies may lead to erroneous statements concerning the importance of exogenous factors in controlling annual reproductive cycles. Although photoperiod has been credited as the dominant environmental regulatory factor in many previous investigations, photoperiodism seems to be temperature sensitive in a majority of the teleost species studied. Responsiveness to exogenous factors appears to depend on the gonadal condition of the beginning controls (effects vary with season). Considerable variation is seen in the reproductive timing mechanisms among the teleosts and few generalizations can be made with regard to phylogenetic, geographic or ecological relationships of the fishes studied.     

Morphological and physiological correlates of unilateral agenesis of the urogenital system in young ovariectomized ACI rats.

ACI rats are distinguished by a polygenic trait resulting in unilateral agenesis of the urogenital system in 20-30% of animals of both sexes. This report details additional features, both morphological and physiological, which distinguish ACI rats with unilateral agenesis of the reproductive and urinary tracts from the majority of ACI rats that have intact urogenital systems. Young female ACI rats were ovariectomized at 34 days of age and sacrificed 19 days later. A preliminary determination of the urogenital morphology was made at the time of ovariectomy and then confirmed by careful abdominal inspection at necropsy. Data on the time of vaginal opening were obtained at selected intervals prior to sacrifice. At necropsy, the mammary glands were removed and were prepared as stained whole mounts for morphological evaluation; the remaining portions of the reproductive tracts were excised, weighed, fixed, and sectioned for microscopic examination. A majority of animals with unilateral agenesis had mammary glands that had higher degrees of glandular proliferation than the mammary glands of intact rats. Unilateral agenesis animals also possessed significantly thicker and heavier uterine horns, despite having been ovariectomized. Furthermore, rats with unilateral agenesis were found to have an earlier time of vaginal opening than that of their intact counterparts. These features of ovariectomized ACI rats with unilateral agenesis are consistent with an active, extra-ovarian source of endogenous estrogen. Further investigation of the endocrinological state of animals with unilateral agenesis of the urogenital tract is warranted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blocking of β-adrenergic receptors during the subfertile period inhibits spontaneous ovarian cyst formation in rats

As aging proceeds, fertility problems arise, and the success rate of in vitro fertilization declines. During reproductive aging, rat ovaries present spontaneous formation of cysts, followed by a concomitant increase in sympathetic nerve activity, causing infertility and cessation of ovarian function. β2-Adrenergic receptors, which are activated by noradrenaline (NA), modify follicular development and steroid secretions; thus, increased nerve activity has been associated with the development and maintenance of cystic structures. The purpose of this work was to block the effect of this sympathetic activity through in vivo administration of propranolol (a β-adrenergic receptor antagonist) to determine whether it delays cyst formation and cessation of the ovarian function in rats that had reached the subfertile period. Propranolol was administrated daily to 8- and 10-month-old rats for 2 months. Estrous cycling activity was monitored by vaginal smear, serum concentration of the steroidal hormones was determined by enzyme-immune assay and morphological analysis of the ovaries was performed using 6 μm tissue slices stained with hematoxylin-eosin. Propranolol increased the number of healthy follicles, the ovulation rate, and levels of serum sexual steroids (androstenedione, testosterone, and estradiol) and recovered estrous cycling activity. It also decreased the number of follicular cysts. These results suggest that the blockade of β-adrenergic receptors recovered ovarian function during reproductive aging. It is suggested that propranolol induces a time-dependent extension of the subfertile window, and it could be used to increase the success rate of fertility programs in aging woman.     

Effect of a 20 kHz sawtooth magnetic field exposure on the estrous cycle in mice

Female mice post weaning were exposed to 20 kHz sawtooth electric and magnetic fields (EMF) with 6.25 microT peak intensity for 6 weeks. Estrous cycles were checked using vaginal smears over the last 10 days of the experimental period. The vaginal smears from EMF-exposed mice revealed an increase in the frequency of one or two phases persisting. The number of estrous cycles less than 1 was more in the EMF-exposed group than in the sham control group. Furthermore, in the EMF-exposed group, the duration of proestrous and metestrous stages of the estrous cycle was significantly increased compared with the control group. In conclusion, our results suggest that exposure to 20 kHz sawtooth EMF may affect normal cycling of the estrous cycle by disrupting the female reproductive endocrine physiology. We should not disregard the possible adverse reproductive effect of the 20 kHz sawtooth EMF generated under the occupational exposure situation in females.