Reproductive Function In Female Mice Research Articles

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

OR09-01 Mini-Puberty Could Be A Key Phase Regulating Reproductive Function In Female Mice

Abstract Disclosure: M. Chester: None. M. Devillers: None. L. Naule: None. F. Souaré: None. R. Corre: None. D. Quintas: None. C. Petrovic: None. J. Cohen-Tannoudji: None. S. Mhaouty-Kodja: None. C.J. Guigon: None. After birth, there is a transient activation of the hypothalamic-pituitary-ovary (HPO) axis, with high secretion of LH and FSH acting on the ovary to promote the secretion of sex steroids and in particular that of estradiol (E2). This period, called mini-puberty, occurs between 10-17 days postnatal (dpn) in mice. During reproductive life, E2 plays an important role on the HPO axis by regulating GnRH pulsatility and, thus, the cyclic fluctuations of gonadotropin hormone levels required for follicular development and ovulation. E2 acts on hypothalamic kisspeptin neurons of the arcuate (ARC) and the anteroventral periventricular nucleus (AVPV), which project to GnRH neurons and regulate their activity by negative and positive feedbacks, respectively. The possibility that E2 contributes, during mini-puberty, to the maturation of hypothalamic structures involved in puberty onset and reproductive function, has never been investigated. The aim of this study was to elucidate the putative roles of mini-puberty on reproduction. We developed a mouse model injected with a GnRH receptor antagonist (GonadoSTOP mice) suppressing LH and FSH surges to cause a 50% decrease in E2 levels during mini-puberty. Our results show that GonadoSTOP mice exhibited later first diestrus 2 (D2) (31.0 dpn versus 29.9 dpn in CTR; n=55-56 mice/group; P=0.0285), suggesting a possible delay in the timing of puberty. During reproductive life, GonadoSTOP mice spent a longer % of time in D2 than control mice, with no alteration in total estrous cycle time (64.4% versus 56% in CTR; n=18-19 mice/group; P=0.0350). Immunohistochemical analysis of kisspeptin neurons in ARC and AVPV of ovariectomized and hormonally-primed mice showed that their numbers were significantly lower in AVPV from GonadoSTOP mice than in control mice (n=5-6 brains/group; P=0.038). In fertility studies, we observed no difference in the time to conception or % of pregnant mice between the two groups at early reproductive life (3, 4 and 5 months). Contrary to these results, at the time of reproductive decline in control mice (11 months), a substantial % of GonadoSTOP mice could still be pregnant (47% versus 18% of CTR mice; P=0.0017). At this age, GonadoSTOP mice had lower LH levels than control mice (P=0.0074), but similar levels to young control mice at 4 months (P>0.05). RT-qPCR analyses of markers reflecting ovarian activity (Cyp19a1, Inhbb, Amh, Gdf9) at 4 and 11 months revealed that ovarian aging occurred similarly in both groups, suggesting that reproductive longevity in GonadoSTOP mice does not have an ovarian origin. Taken together these data suggest that transient and sustained activation of the HPO axis at mini-puberty may regulate key aspects of reproductive function with long-term impact on fertility, possibly by acting on the hypothalamus. Presentation: Friday, June 16, 2023

The role of minipuberty on reproductive function in female mice

The role of minipuberty on reproductive function in female mice

Persistent follicular granulosa cell senescence and apoptosis induced by methotrexate leading to oocyte dysfunction and aberrant embryo development.

Evidence from clinical cases indicates an association between the low success rate of in vitro fertilization (IVF) and ovarian injury due to previous methotrexate (MTX) administration. Therefore, it is necessary to develop and propose reasonable clinical drug guidelines to improve the quality of oocytes and the development of embryos before pregnancy. In this study, we established a mouse model with previous MTX exposure to validate the effects of MTX on reproductive function in female mice. We observed that MTX administration could result in a decrease in the success rate of fertilization and an aberrant embryonic development in both natural fertilization and IVF, even after completion of five to six ovulation cycles after MTX withdrawal. Further research revealed senescence and apoptosis of follicular granulosa cells (GCs), accompanied by arrested follicle development and aberrant estradiol and anti‐Mullerian hormone levels. Supportive evidence indicated that MTX administration induced senescence and apoptosis of human GCs in vitro, and the effects were consistent with the high levels of p21, p53, and oxidative stress. We further demonstrated that folic acid (FA) could improve oocyte function and embryonic development in vivo and in vitro by protecting GCs against apoptosis and senescence. Based on these findings, we propose the implementation of extended intervals between MTX exposure and conception or IVF and recommend FA as a special dietary supplement during this interval period; however, prospective inquiry in humans is necessary to further understand the relationship between MTX and FA recovery.

MiR-29a/b1 Regulates the Luteinizing Hormone Secretion and Affects Mouse Ovulation

miR-29a/b1 was reportedly involved in the regulation of the reproductive function in female mice, but the underlying molecular mechanisms are not clear. In this study, female mice lacking miR-29a/b1 showed a delay in vaginal opening, irregular estrous cycles, ovulation disorder and subfertility. The level of luteinizing hormone (LH) was significantly lower in plasma but higher in pituitary of mutant mice. However, egg development was normal in mutant mice and the ovulation disorder could be rescued by the superovulation treatment. These results suggested that the LH secretion was impaired in mutant mice. Further studies showed that deficiency of miR-29a/b1 in mice resulted in an abnormal expression of a number of proteins involved in vesicular transport and exocytosis in the pituitary, indicating the mutant mice had insufficient LH secretion. However, the detailed mechanism needs more research.

Male mice ultrasonic vocalizations enhance female sexual approach and hypothalamic kisspeptin neuron activity

Vocal communication in animals is important for ensuring reproductive success. Male mice emit song-like “ultrasonic vocalizations (USVs)” when they encounter female mice, and females show approach to the USVs. However, it is unclear whether USVs of male mice trigger female behavioral and endocrine responses in reproduction. In this study, we first investigated the relationship between the number of deliveries in breeding pairs for 4months and USVs syllables emitted from those paired males during 3min of sexual encounter with unfamiliar female mice. There was a positive correlation between these two indices, which suggests that breeding pairs in which males could emit USVs more frequently had more offspring. Further, we examined the effect of USVs of male mice on female sexual behavior. Female mice showed more approach behavior towards vocalizing males than devocalized males. Finally, to determine whether USVs of male mice could activate the neural system governing reproductive function in female mice, the activation of kisspeptin neurons, key neurons to drive gonadotropin-releasing hormone neurons in the hypothalamus, was examined using dual-label immunocytochemistry with cAMP response element-binding protein phosphorylation (pCREB). In the arcuate nucleus (Arc), the number of kisspeptin neurons expressing pCREB significantly increased after exposure to USVs of male as compared with noise exposure group. In conclusion, our results suggest that USVs of male mice promote fertility in female mice by activating both their approaching behavior and central kisspeptin neurons.

Evaluation of Neonatal Exposure to Mycoestrogens Zearalenone and Alpha-zearalenol on Puberty and Reproductive Function in Female Mice

Evaluation of Neonatal Exposure to Mycoestrogens Zearalenone and Alpha-zearalenol on Puberty and Reproductive Function in Female Mice

Developmental Exposure to Ethinylestradiol Affects Reproductive Physiology, the GnRH Neuroendocrine Network and Behaviors in Female Mouse.

During development, environmental estrogens are able to induce an estrogen mimetic action that may interfere with endocrine and neuroendocrine systems. The present study investigated the effects on the reproductive function in female mice following developmental exposure to pharmaceutical ethinylestradiol (EE2), the most widespread and potent synthetic steroid present in aquatic environments. EE2 was administrated in drinking water at environmentally relevant (ENVIR) or pharmacological (PHARMACO) doses [0.1 and 1 μg/kg (body weight)/day respectively], from embryonic day 10 until postnatal day 40. Our results show that both groups of EE2-exposed females had advanced vaginal opening and shorter estrus cycles, but a normal fertility rate compared to CONTROL females. The hypothalamic population of GnRH neurons was affected by EE2 exposure with a significant increase in the number of perikarya in the preoptic area of the PHARMACO group and a modification in their distribution in the ENVIR group, both associated with a marked decrease in GnRH fibers immunoreactivity in the median eminence. In EE2-exposed females, behavioral tests highlighted a disturbed maternal behavior, a higher lordosis response, a lack of discrimination between gonad-intact and castrated males in sexually experienced females, and an increased anxiety-related behavior. Altogether, these results put emphasis on the high sensitivity of sexually dimorphic behaviors and neuroendocrine circuits to disruptive effects of EDCs.

The effects of in utero bisphenol A exposure on reproductive capacity in several generations of mice

In utero bisphenol A (BPA) exposure affects reproductive function in the first generation (F1) of mice; however, not many studies have examined the reproductive effects of BPA exposure on subsequent generations. In this study, pregnant mice (F0) were orally dosed with vehicle, BPA (0.5, 20, and 50μg/kg/day) or diethylstilbestrol (DES; 0.05μg/kg/day) daily from gestation day 11 until birth. F1 females were used to generate the F2 generation, and F2 females were used to generate the F3 generation. Breeding studies at the ages of 3, 6, and 9months were conducted to evaluate reproductive capacity over time. Further, studies were conducted to evaluate pubertal onset, litter size, and percentage of dead pups; and to calculate pregnancy rate, and mating, fertility, and gestational indices. The results indicate that BPA exposure (0.5 and 50μg/kg/day) significantly delayed the age at vaginal opening in the F3 generation compared to vehicle control. Both DES (0.05μg/kg/day) and BPA (50μg/kg/day) significantly delayed the age at first estrus in the F3 generation compared to vehicle control. BPA exposure reduced gestational index in the F1 and F2 generations compared to control. Further, BPA exposure (0.5μg/kg/day) compromised the fertility index in the F3 generation compared to control. Finally, in utero BPA exposure reduced the ability of female mice to maintain pregnancies as they aged. Collectively, these data suggest that BPA exposure affects reproductive function in female mice and that some effects may be transgenerational in nature.

Neurokinin B is critical for normal timing of sexual maturation but dispensable for adult reproductive function in female mice.

Humans carrying mutations in neurokinin B (NKB) or the NKB receptor fail to undergo puberty due to decreased secretion of GnRH. Despite this pubertal delay, many of these patients go on to achieve activation of their hypothalamic-pituitary-gonadal axis in adulthood, a phenomenon termed reversal, indicating that NKB signaling may play a more critical role for the timing of pubertal development than adult reproductive function. NKB receptor-deficient mice are hypogonadotropic but have no defects in the timing of sexual maturation. The current study has performed the first phenotypic evaluation of mice bearing mutations in Tac2, the gene encoding the NKB ligand, to determine whether they have impaired sexual development similar to their human counterparts. Male Tac2-/- mice showed no difference in the timing of sexual maturation or fertility compared with wild-type littermates and were fertile. In contrast, Tac2-/- females had profound delays in sexual maturation, with time to vaginal opening and first estrus occurring significantly later than controls, and initial abnormalities in estrous cycles. However, cycling recovered in adulthood and Tac2-/- females were fertile, although they produced fewer pups per litter. Thus, female Tac2-/- mice parallel humans harboring NKB pathway mutations, with delayed sexual maturation and activation of the reproductive cascade later in life. Moreover, direct comparison of NKB ligand and receptor-deficient females confirmed that only NKB ligand-deficient animals have delayed sexual maturation, suggesting that in the absence of the NKB receptor, NKB may regulate the timing of sexual maturation through other tachykinin receptors.

Conditional knockout of the androgen receptor in gonadotropes reveals crucial roles for androgen in gonadotropin synthesis and surge in female mice.

Polycystic ovary syndrome is the major cause of infertility in reproductive aged women. Polycystic ovary syndrome is associated with high circulating levels of androgens and impaired metabolic function. The goal of this study was to understand how androgen signaling via the androgen receptor (AR) affects reproductive function. We knocked out the AR gene specifically in pituitary gonadotropes (PitARKO) to explore the role of androgen on the development of reproductive function in female mice. There was no difference in the age of puberty between control and PitARKO littermates, which was assessed by the ages of vaginal opening and first estrus. Cyclicity and fertility were also studied, and there was no significant difference between control and PitARKO mice. We observed a significant decrease in basal FSH serum and mRNA levels with no corresponding change in LH serum and mRNA levels. Although the numbers of litters born to control and PitARKO females were the same, the litter size was significantly smaller for PitARKO mice. The LH and FSH responses to ovariectomy was altered with reduced LH/FSH hormone and mRNA levels in PitARKO females. This reduction may be due to reduced expression of activin A/B and gnrhr. The preovulatory surge levels of LH and FSH were dramatically lower in PitARKO mice. The number of corpora lutea was decreased whereas the number of antral follicles was similar between control and PitARKO mice. Overall the pituitary AR contributes to the elaboration of the LH surge and normal reproductive function by regulating LH/FSH expression and secretion.

Instillation of diesel exhaust particles on the posterior wall of pharynx on reproductive function in female mice

The present work aims to investigate the effects of subacute exposure to diesel exhaust particles (DEP) on reproductive function in female mice. A total of 168 ICR (Institute of Cancer Research) mice were randomly divided into four groups by numeration table method, including the low (B), middle (C), high (D) dose DEP exposure groups and the control group (A). Each group consisted of 42 mice. Mice were inoculated with 30 µl DEP suspension at 0.8 (B), 3.0 (C), 12.0 (D) µg/µl, respectively, or the same volume of phosphate-buffered saline (A) on pharynx posterior wall per triduum for 4 times. The body weight and ovary weight were tested and ovary weight/body weight ratios were calculated. Rates of survival, germinal vesicle breakdown, extrusion of the first polar body, in-vitro fertilization and quantity of mitochondrial DNA for the oocytes were investigated. Ultrastructural changes of the oocytes were observed. The ovary weight/body weight ratios were (15.4 ± 7.3) × 10(-5), (14.1 ± 6.8) × 10(-5), (8.2 ± 0.7) × 10(-5) and (7.2 ± 2.5) × 10(-5) in groups A, B, C and D (F = 3.841, P < 0.05). In groups A, B, C and D at 48 h post-insemination, rates of oocyte survival were 64.3%, 56.8%, 39.5% and 32.9% (χ(2) = 21.575, P < 0.05), rates of extrusion of the first polar body were 75.5%, 65.3%, 37.0% and 27.1% (χ(2) = 52.772, P < 0.05), rates of 2-cell embryos were 27.9%, 39.1%, 17.6% and 12.5% (χ(2) = 20.148, P < 0.05), and rates of embryos over 2 cells were 45.3%, 32.2%, 12.5% and 13.9% (χ(2) = 32.135, P < 0.05), respectively, and were significantly lower in groups C and D compared with group A (P < 0.05). Logarithmic values of mitochondrial DNA copy numbers were 6.54 ± 0.13, 6.48 ± 0.09, 5.57 ± 0.15 and 5.41 ± 0.07 in groups A, B, C and D, respectively, and were significantly lower in groups C and D compared with group A or B (F = 89.241, P < 0.05). A number of mitochondria of the oocytes exhibited tremendous tumescence and vacuolization in groups C and D, which was contrast to a roughly normal appearance in groups A and B. DEP is noxious to murine female reproduction. Subacute exposure to DEP injures the ovary and oocyte resulting in compromised ovarian function and fertilizability of the oocyte.

Role of Central Nervous System and Ovarian Insulin Receptor Substrate 2 Signaling in Female Reproductive Function in the Mouse1

Insulin receptor signaling regulates female reproductive function acting in the central nervous system and ovary. Female mice that globally lack insulin receptor substrate (IRS) 2, which is a key mediator of insulin receptor action, are infertile with defects in hypothalamic and ovarian functions. To unravel the tissue-specific roles of IRS2, we examined reproductive function in female mice that lack Irs2 only in the neurons. Surprisingly, these animals had minimal defects in pituitary and ovarian hormone levels, ovarian anatomy and function, and breeding performance, which indicates that the central nervous system IRS2 is not an obligatory signaling component for the regulation of reproductive function. Therefore, we undertook a detailed analysis of ovarian function in a novel Irs2 global null mouse line. Comparative morphometric analysis showed reduced follicle size, increased numbers of atretic follicles, as well as impaired oocyte growth and antral cavity development in Irs2 null ovaries. Granulosa cell proliferation was also defective in the Irs2 null ovaries. Furthermore, the insulin- and eCG-stimulated phosphoinositide-3-OH kinase signaling events, which included phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3-beta, were impaired, whereas mitogen-activated protein kinase signaling was preserved in Irs2 null ovaries. These abnormalities were associated with reduced expression of cyclin D2 and increased CDKN1B levels, which indicates dysregulation of key components of the cell cycle apparatus implicated in ovarian function. Our data suggest that ovarian rather than central nervous system IRS2 signaling is important in the regulation of female reproductive function.

Null Mutation in Transforming Growth Factor β1 Disrupts Ovarian Function and Causes Oocyte Incompetence and Early Embryo Arrest

TGFbeta1 is implicated in regulation of ovarian function and the events of early pregnancy. We have investigated the effect of null mutation in the Tgfbeta1 gene on reproductive function in female mice. The reproductive capacity of TGFbeta1 null mutant females was severely impaired, leading to almost complete infertility. Onset of sexual maturity was delayed, after which ovarian function was disrupted, with extended ovarian cycles, irregular ovulation, and a 40% reduction in oocytes ovulated. Serum FSH and estrogen content were normal, but TGFbeta1 null mutant mice failed to display the characteristic proestrus surge in circulating LH. Ovarian hyperstimulation with exogenous gonadotropins elicited normal ovulation rates in TGFbeta1 null mutant mice. After mating with wild-type stud males, serum progesterone content was reduced by 75% associated with altered ovarian expression of mRNAs encoding steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase-1 and P450 17 alpha-hydroxylase/C17-20-lyase. Embryos recovered from TGFbeta1 null mutant females were developmentally arrested in the morula stage and rarely progressed to blastocysts. Attempts to rescue embryos by exogenous progesterone administration and in vitro culture were unsuccessful, and in vitro fertilization and culture experiments demonstrated that impaired development is unlikely to result from lack of maternal tract TGFbeta1. We conclude that embryo arrest is due to developmental incompetence in oocytes developed in a TGFbeta1-deficient follicular environment. This study demonstrates that TGFbeta1 is a critical determinant of normal ovarian function, operating through regulation of LH activity and generation of oocytes competent for embryonic development and successful initiation of pregnancy.

Reproductive Function in Female Mice Lacking the Gene for Endothelial Nitric Oxide Synthase

Nitric oxide (NO) acts as a neuronal messenger in both the central and peripheral nervous systems and has been implicated in reproductive physiology and behavior. Pharmacological inhibition of nitric oxide synthase (NOS) with the nonspecific NOS inhibitor, l-NG-nitro-Arg-methyl ester (l-NAME), induced deficits in both the number of ovarian rupture sites and the number of oocytes recovered in the oviducts of mice. Female neuronal NOS knockout (nNOS−/−) mice have normal numbers of rupture sites, but reduced numbers of oocytes recovered following systemic injections of gonadotropins, suggesting that NO produced by nNOS accounts, in part, for deficits in ovulatory efficiency observed after l-NAME administration. Additionally, endothelial NOS knockout (eNOS−/−) mice have reduced numbers of ovulated oocytes after superovulation. Because endothelial NOS has been identified in ovarian follicles, and because of the noted reduced breeding efficiency of eNOS−/− mice, the present study sought to determine the role of NO from eNOS in mediating the number of rupture sites present after ovulation. Estrous cycle length and variability were consistently reduced in eNOS−/− females. The number of rupture sites was normal in eNOS−/− mice under natural conditions and after administration of exogenous GnRH. After exogenous gonadotropin administration, eNOS−/− females displayed a significant reduction in the number of ovarian rupture sites. Female eNOS−/− mice also produced fewer pups/litter compared to WT mice. These data suggest that NO from endothelial sources might play a role in mediating rodent ovulation and may be involved in regulation of the timing of the estrous cycle.

Decline in male mouse pheromone with age.

An age-related decline in urinary-borne pheromone was found in male C57BL/6J mice aged from 2 to 30 months. Pheromone activity, estimated by bioassay, declined sharply after about 10 months of age. Two other strains of mice tested (DBA/2J and CBA/HT6J) also appeared to show an age-related decline in pheromone activity. Within each strain, however, pheromone activity was consistently similar to or higher than that of the C57BL/6J male mice. The DBA/2J and BALB/cWt strains appeared to be high pheromone producers, and the C57BL/6J and CBA/HT6J strains, low producers. This report is the first demonstration of a decline with age in male mouse pheromone activity. This decline appears to be synchronized with the well-defined loss of reproductive function in female mice.