Mating-induced Increase Research Articles

Mating-induced increase of kynurenine in Drosophila ovary enhances starvation resistance of progeny

The maternal nutritional environment can impact progeny development, stress tolerance, and longevity. Such phenotypic variation of offspring resulting from the maternal environment is often referred to as the 'maternal effect' and is observed across taxa, including in humans. While some mechanisms behind maternal effects have been revealed, such as histone modification, many studies rely on drastic genetic or nutritional manipulation in describing these mechanisms. Here we aimed to reveal how the maternal environment is regulated under physiological conditions to affect the progeny. Specifically, we detailed metabolic regulation in oocytes in response to mating using Drosophila melanogaster fruit flies. Using liquid chromatography–mass spectrometry (LC-MS), we found that upon mating, the ovary metabolites shifted, predominantly toward increasing amino acids (AAs) and the tryptophan/kynurenine (Trp/Kyn) pathway. This mating-induced increase in ovary Kyn was driven by increased Kyn production in the fat body (FB), a functional counterpart of the mammalian liver and white adipose tissue, and the source of Kyn storage for the ovary after mating. Furthermore, we show maternal Kyn repression decreased the starvation resistance of progeny, and that administering exogenous Kyn to the maternal generation enhanced the starvation resistance of female progeny. Taken together, these findings point to a previously unidentified role of FB Kyn distribution during reproduction on progeny survival.

Regulation of Mating-Induced Increase in Female Germline Stem Cells in the Fruit Fly Drosophila melanogaster

In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. We have extensively studied mating-induced increase in female GSCs of D. melanogaster at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model. We also discuss the remaining issues that should be addressed in the future.

Mating-induced increase in <i>Kiss1</i> mRNA expression in the anteroventral periventricular nucleus prior to an increase in LH and testosterone release in male rats

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

Quantification of in vivo progenitor mutation accrual with ultra-low error rate and minimal input DNA using SIP-HAVA-seq.

Assaying in vivo accrual of DNA damage and DNA mutations by stem cells and pinpointing sources of damage and mutations would further our understanding of aging and carcinogenesis. Two main hurdles must be overcome. First, in vivo mutation rates are orders of magnitude lower than raw sequencing error rates. Second, stem cells are vastly outnumbered by differentiated cells, which have a higher mutation rate—quantification of stem cell DNA damage and DNA mutations is thus best performed from small, well-defined cell populations. Here we report a mutation detection technique, based on the “duplex sequencing” principle, with an error rate below ∼10−10 and that can start from as little as 50 pg DNA. We validate this technique, which we call SIP-HAVA-seq, by characterizing Caenorhabditis elegans germline stem cell mutation accrual and asking how mating affects that accrual. We find that a moderate mating-induced increase in cell cycling correlates with a dramatic increase in accrual of mutations. Intriguingly, these mutations consist chiefly of deletions in nonexpressed genes. This contrasts with results derived from mutation accumulation lines and suggests that mutation spectrum and genome distribution change with replicative age, chronological age, cell differentiation state, and/or overall worm physiological state. We also identify single-stranded gaps as plausible deletion precursors, providing a starting point to identify the molecular mechanisms of mutagenesis that are most active. SIP-HAVA-seq provides the first direct, genome-wide measurements of in vivo mutation accrual in stem cells and will enable further characterization of underlying mechanisms and their dependence on age and cell state.

Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

AMPA receptors in the medial amygdala are critical for establishing a neuroendocrine memory in the female rat

We sought to examine AMPA receptor (AMPAR) function in the medial posterodorsal amygdala (MePD), as glutamate neurotransmission is critical for the neural response to vaginal-cervical stimulation that initiates pregnancy or pseudopregnancy. Female rats were infused with the AMPAR antagonist CNQX or vehicle directly into the MePD via bilaterally implanted cannulae, then either returned to their homecage (HC), or received 15 mounts-without-intromissions (MO) or 15 intromissions (15I) from a male. Expression of the activity marker EGR-1 was used to determine the CNQX concentration which would prevent mating-induced activation of MePD neurons. Separate cannulated females received CNQX infusions into the MePD prior to receiving 15I, and the oestrous cycle length was monitored by daily vaginal lavages. Infusion of CNQX (500 nm) blocked mating-induced neural activation and lengthened the oestrous cycle, demonstrating AMPAR involvement in the formation of pseudopregnancy. To further explore this involvement, separate groups of 15I, MO and HC females were killed 90 min or 3 h after testing treatment. Brain sections were immunolabeled for AMPAR-subunit GluR1 phosphorylated at one of two sites (Serine-831 or Serine-845), or total GluR1 and GluR2, and immunofluorescence intensity was measured in the MePD, hippocampus and hypothalamus. A mating-induced increase in Serine-831 phosphorylation after 3 h was observed only in the MePD, whereas there was no effect on Serine-845 phosphorylation. Additionally, we observed a time-dependent increase in total GluR1 staining intensity. These results suggest an increased AMPAR function in the MePD after receipt of VCS, and a role for AMPAR in the neural response to VCS resulting in pseudopregnancy.

Mating increases starvation resistance and decreases oxidative stress resistance in Drosophila melanogaster females

Mating stimulates complex physiological changes in females of Drosophila melanogaster. Long-term effects of mating are manifested in increased fecundity and shortened lifespan. It is not clear how mating affects stress resistance in fly females. We addressed this question here and found that mated and highly fecund wild-type D. melanogaster females have significantly higher resistance to starvation throughout their lifetime than age-matched virgin females. Mean survival time under starvation was age dependent with maximum survival time observed in 15-day-old mated females. Mating-induced increase in starvation resistance was associated with significantly higher fat reserves stored as triacylglycerols. While mated females had higher resistance to starvation, their resistance to oxidative stress was significantly lower than in age-matched virgins. Our study revealed that mating leads to an opposing relationship between resistance to starvation and resistance to oxidative stress in Drosophila females. Thus, shortened lifespan of mated females is associated with their high-fat content and greater susceptibility to oxidative stress.

Allatotropin-like peptide inHeliothis virescens: Tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.

Temporal changes occur in the neuroendocrine control of gonadotropin secretion in aging female rats: role of progesterone.

The present study examined the gonadotropin surge-inducing actions of estradiol (E(2)), both alone and with progesterone (P(4)), in middle-aged, early persistent-estrous (PE) female rats that had become PE within 35 days. In addition, we also assessed the effect of P(4) on the mating-induced gonadotropin surges in these acyclic animals. Early PE rats were ovariectomized and received E(2) implants (Day 0). On Day 4, an s.c. injection of P(4) (0.5 mg/ 100 g body weight) at 1200 h markedly increased plasma P(4) and elicited both LH and FSH surges, whereas vehicle-treated controls displayed no rise in P(4) or gonadotropins. This observation confirms that at middle age, female rats no longer respond to the positive-feedback stimulation of E(2) on gonadotropin surges whenever the estrous cyclicity ceases. As PE continued, such a surge-inducing action of E(2) plus P(4) became diminished after 75 days of PE and disappeared thereafter. When caged with males, vehicle-treated early PE rats display a mating-induced increase in P(4) from the adrenal along with small gonadotropin surges. The amplitude of these mating-induced gonadotropin surges was enhanced by supplementation with exogenous P(4) in early PE rats. Our findings indicate that during the early phase of PE, the surge-inducing action of E(2) and P(4) remains intact but deteriorates as PE continues. Thus, a deficiency in P(4) secretion during aging may contribute to the diminished gonadotropin surge response in the hypothalamic-pituitary axis and the subsequent cessation of estrous cyclicity.

Neuronal activation in the caudal brainstem associated with mating by voles

The expression of c-fos, a marker of neuronal activation, was examined in the gracile nucleus (GN) and nucleus of the solitary tract (NTS) after social interactions, including mating, between male and female prairie voles. In GN, mating, but not non-sexual interactions, induced similar significant increases in c-fos immunoreactivity in both males and females. The increased immunoreactivity was concentrated in medial and dorsal GN suggesting that expression was driven by stimulation of reproductive organs. In contrast, in NTS, mating-induced increases in c-fos expression occurred only in males. These results suggest that both GN and NTS comprise different functional components of mating circuitry and may contribute to pair bonding in monogamous voles.

Somatosensory stimuli evoke norepinephrine release in the anterior ventromedial hypothalamus of sexually receptive female rats.

We used in vivo brain microdialysis to determine the role of specific copulatory stimuli in mating-induced release of norepinephrine in the lateral ventromedial hypothalamus (VMH) of hormone-treated, sexually receptive female rats. Ovariectomized rats implanted with a unilateral guide cannula aimed at the ventrolateral VMH received systemic injections of estradiol benzoate daily for 2 days before and progesterone 4 h before the initiation of a 1-h behavioural test. Dialysis probes were lowered immediately after progesterone administration, and 20-min dialysis samples were collected until 1 h after the termination of behavioural testing. Norepinephrine content of dialysates was quantified by high performance liquid chromatography with electrochemical detection. During mating tests with male rats, dialysate levels of norepinephrine increased significantly over baseline in sexually receptive females with probe placements in the anterior but not posterior VMH. Norepinephrine levels were unchanged if rats were nonreceptive, even if males mounted vigorously and probes were located in the anterior VMH. Hormone-treated females that were placed on male-soiled bedding for 1 h showed no changes in dialysate levels of norepinephrine. Similarly, females in which vaginocervical stimulation was prevented by a vaginal mask failed to show increased levels of norepinephrine in dialysates collected from the anterior VMH, even if they displayed high levels of lordosis behaviour. Thus, the release of norepinephrine is not a result of executing the lordosis posture. The findings suggest that mating-induced increases in norepinephrine release in hormone-treated, sexually receptive rats are confined to the anterior VMH and that somatosensory rather than chemosensory stimuli evoke norepinephrine release. Moreover, experiments with vaginal masks indicate that vaginocervical stimulation is necessary for mating-evoked norepinephrine release in the anterior VMH.

Effects of paced and non-paced mating stimulation on plasma progesterone, 3 alpha-diol and corticosterone.

In the female rat, gonadal and adrenal progestins and androgens modulate sexual receptivity and in turn, their levels increase in response to mating stimulation. Paced mating, in which the female controls the timing of sexual contacts with the male, is particularly effective at eliciting acute increases in progesterone (P) and 5 alpha-Androstane-3 alpha, 17 beta-diol (3 alpha-Diol). Interestingly, restraint stress produces comparable increases in P and 3 alpha-Diol levels, as well as increases in corticosterone (CORT) levels. In this study, we explored the possibility that paced mating would be associated with increased CORT, in conjunction with mating-induced increases in P and 3 alpha-Diol. Ovariectomized rats primed with estradiol benzoate (10 micrograms in oil SC) and P (0.5 mg in oil s.c.) received a single ejaculatory series from males in paced or non-paced mating tests. Fifteen minutes post-mating rats were exposed to CO2 and rapidly decapitated for the collection of trunk blood and determination of P, 3 alpha-Diol and CORT via radioimmunoassay. As expected, P and 3 alpha-Diol concentrations were significantly elevated in serum obtained from animals allowed to pace their sexual contacts with males compared to those which did not pace their contacts. Importantly, although all mated animals had CORT levels between 10-20 micrograms/dl, there were no differences between paced and non-paced conditions. This suggests that the acute rises in P and 3 alpha-Diol in response to paced mating are not due to paced mating being more stressful than non-paced mating.

Effect of mating on plasma juvenile hormone esterase activity in females ofChoristoneura fumiferana andC. rosaceana

We measured juvenile hormone esterase (JHE) activity in the plasma of virgin and mated Choristoneura fumiferana and C. rosaceana female moths. In these two species, JH III acid was the only measurable product of in vitro JH III degradation in the plasma. Rates of JH III ester hydrolysis were generally higher in C. rosaceana than in C. fumiferana, but followed a similar pattern in both species, with high activity measured on day 7 of the pupal stage, the lowest activity measured at emergence, and a subsequent increase observed between days 0 and 1. Mating on day 0, in both species, brought about a significant increase in JHE activity on day 1, relative to virgins. On days 3 and 5, rates of JH degradation did not differ between virgin and mated females. The mating-induced increase in JHE activity on day 1 was not the result of a decrease in blood volume in mated females relative to virgins. Fluctuations in JHE activity did not appear to depend on changes in plasma protein concentration. In both species, topical application of JH I or methoprene, on the day of emergence, resulted in dose- and time-dependent increases in JHE activity relative to controls. © 1996 Wiley-Liss, Inc.

Patterns of induction of the immediate-early genes c-fos and egr-1 in the female rat brain following differential amounts of mating stimulation.

Vaginocervical stimulation received either during mating or by artificial mechanical means has been shown to induce FOS expression in medial amygdala, preoptic area, hypothalamus, and midbrain of female rats. While mating-induced increases in FOS-like immunoreactivity (FOS-IR) have been shown to require intromissive stimulation from males, the pattern of FOS-IR in animals receiving numbers of intromissions across a range relevant to the induction of the prolactin surges of early pregnancy has not been explored. Experiment 1 examined brain FOS-IR following 15 mounts without intromission or 5, 10, or 15 intromissions in ovariectomized females treated with estrogen and progesterone; these treatments are known to be less than or more than sufficient to trigger prolactin surges in cycling females. FOS was expressed in a graded fashion in the medial amygdala with respect to the numbers of intromissions received and in an all-or-nothing manner in preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus of the hypothalamus. In experiment 2, 15 intromissions induced expression of another immediate-early gene, egr-1, in each of these same areas as well as in a second division of the bed nucleus of the stria terminalis and in the paraventricular nucleus of the hypothalamus. These studies demonstrate that mating is differentially effective in inducing FOS expression in responsive brain areas and point to the medial amygdala as a site in which summation of intromissive stimulation may occur. Furthermore, the induction of EGR-1 may be a more sensitive marker for mating-induced neural activation in these areas than is FOS.

The role of calcium in the Chlamydomonas reinhardtii mating reaction.

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.

Mating-induced increases in FOS protein in preoptic area and medial amygdala of cycling female rats

Genitosensory stimulation received during mating initiates neural and endocrine changes necessary for pregnancy. The present study examined sites of induction of the protoncogene, c- Fos, after mating in the cycling female rat to determine neural sites activated by such stimulation that might be involved in pregnancy initiation. Two groups of cycling female rats were exposed to males on the evening of proestrus and remained with the male until receiving either 14 mounts-with-intromission including ejaculations (Intromissions) or a control number of mounts-without-intromission (Mounts-Only). These two mating treatments were previously shown to induce pregnancy/pseudopregnancy in 100% and 0% of the animals, respectively. Seventy-five minutes after mating, females were perfused intracardially with 2% paraformaldehyde/2.5% acrolein, and the brains were processed for FOS immunocytochemistry using standard procedures. FOS-immunoreactive cells (FOS-IR) were counted in standard template quadrilaterals in the preoptic area (POA), medial amygdala (mAMYG), and the paraventricular (PVN), ventromedial (VMN), and dorsomedial (DMN) nuclei of the hypothalamus at 50× magnification using a camera lucida. Significantly higher numbers of FOS-IR cells were seen in Intromissions females above Mounts-Only females in the POA and mAMYG, demonstrating that c- Fos expression in these areas depended upon cervical-vaginal stimulation rather than on cutaneous somatosensory input received in Mounts-Only tests. The cells within the POA and mAMYG that are activated in response to the intromittive stimulus may be directly involved in pathways triggering one or several of the neuroendocrine responses to mating in the female rat.