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Scientific Reports Oct 2022The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized...
The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized by distinct hormone concentration profiles. Given the difficulty of repeatedly sampling plasma steroid hormones from rodents, the primary method for classifying estrous stage is by identifying vaginal epithelial cell types. However, manual classification of epithelial cell samples is time-intensive and variable, even amongst expert investigators. Here, we use a deep learning approach to achieve classification accuracy at expert level. Due to the heterogeneity and breadth of our input dataset, our deep learning approach ("EstrousNet") is highly generalizable across rodent species, stains, and subjects. The EstrousNet algorithm exploits the temporal dimension of the hormonal cycle by fitting classifications to an archetypal cycle, highlighting possible misclassifications and flagging anestrus phases (e.g., pseudopregnancy). EstrousNet allows for rapid estrous cycle staging, improving the ability of investigators to consider endocrine state in their rodent studies.
Topics: Female; Animals; Rodentia; Deep Learning; Estrus; Estrous Cycle; Hormones
PubMed: 36271290
DOI: 10.1038/s41598-022-22392-w -
The Journal of Reproduction and... Oct 2022The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews,...
The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44-48 h after mating. Another set of embryos was collected 26-27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.
Topics: Animals; Embryo Transfer; Female; Gonadotropins; Humans; Male; Oocytes; Receptor Protein-Tyrosine Kinases; Receptors, Cholinergic; Shrews
PubMed: 36070889
DOI: 10.1262/jrd.2022-003 -
Life (Basel, Switzerland) May 2022Reliable pregnancy diagnostics would be beneficial for monitoring polar bear () populations both in situ and ex situ, but currently there is no method of non-invasive...
Reliable pregnancy diagnostics would be beneficial for monitoring polar bear () populations both in situ and ex situ, but currently there is no method of non-invasive pregnancy detection in this species. Recent reports in several carnivore species described the identification of fecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. The objective of the current analysis was to utilize an unbiased, antibody-free, label-free method for the identification and quantification of fecal proteins to determine if differences associated with pregnancy are detectable in polar bears. Protein was extracted from fecal samples ( = 48) obtained from parturient ( = 6) and non-parturient ( = 6) profiles each at four timepoints: pre-breeding season, embryonic diapause, early placental pregnancy, and mid-placental pregnancy. Protein was prepared and analyzed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system. A total of 312 proteins was identified and quantified; however, coefficients of variation (CV) were high for both abundance ratio variability (384.8 ± 61.0% SEM) and within group variability (86.8 ± 1.5%). Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology's limitations, but rather, are reflective of true variation that exists among samples.
PubMed: 35743827
DOI: 10.3390/life12060796 -
Frontiers in Neuroscience 2022Some of our breeding programs include the use of Prm1 male Homozygous mice which are naturally sterile. This removes the need to use vasectomized males to induce...
Some of our breeding programs include the use of Prm1 male Homozygous mice which are naturally sterile. This removes the need to use vasectomized males to induce pseudopregnancy in female mice. These males can be kept for up to 9 months and are housed with a companion female. During the timed mating period the companion female is replaced with a new female. This procedure can occur at regular intervals causing a significant increase in cage activity; one of our objectives was to determine whether this was as a result of timed mating. We wanted to investigate the disruption caused to mice during the day of the swap and how long it would take for the cage activity to return to pre-replacement baseline levels. We hypothesized that this impact would be reflected as a significant increase in cage activity, which in itself may not be a result of a negative experience but the potential of repeated disruption to their activity pattern should be considered. We used a well-known home-cage monitoring system to assess changes to the activity pattern in cages when a companion female is replaced. Data from our initial study showed that in the 2-h period after the female is replaced there is a significant increase in cage activity compared to the same time frame on the previous day. In the subsequent study, where no cage change occurred, an increase in activity was also observed when females were replaced; this returned to baseline after approximately 4 h. Prolonged activity during the rest period of mice (over 2 h) could lead to them being fatigued during their active period; therefore, as a refinement we propose that timed matings be performed later in the day, at a time when the animals are active.
PubMed: 35281486
DOI: 10.3389/fnins.2022.786652 -
Biomolecules Jan 2022As patients recently diagnosed with T1D and patients with T2D have residual beta cell mass, there is considerable effort in beta cell biology to understand the...
As patients recently diagnosed with T1D and patients with T2D have residual beta cell mass, there is considerable effort in beta cell biology to understand the mechanisms that drive beta cell regeneration as a potential cellular therapy for expanding patients' residual beta cell population. Both mouse and human studies have established that beta cell mass expansion occurs rapidly during pregnancy. To investigate the mechanisms of beta cell mass expansion during pregnancy, we developed a novel in vivo and in vitro models of pseudopregnancy. Our models demonstrate that pseudopregnancy promotes beta cell mass expansion in parous mice, and this expansion is driven by beta cell proliferation rather than hypertrophy. Importantly, estrogen, progesterone, and placental lactogen induce STAT5A signaling in the pseudopregnancy model, demonstrating that this model successfully recapitulates pregnancy-induced beta cell replication. We then created an in vitro model of pseudopregnancy and found that the combination of estrogen and placental lactogen induced beta cell replication in human islets and rat insulinoma cells. Therefore, beta cells both in vitro and in vivo increase proliferation when subjected to the pseudopregnancy cocktail compared to groups treated with estradiol or placental lactogen alone. The pseudopregnancy models described here may help inform novel methods of inducing beta cell replication in patients with diabetes.
Topics: Animals; Cell Division; Female; Humans; Insulin-Secreting Cells; Islets of Langerhans; Mice; Placenta; Placental Lactogen; Pregnancy; Rats
PubMed: 35204716
DOI: 10.3390/biom12020215 -
Reproduction & Fertility Dec 2021Previous reports indicate that red pandas () may experience fetal loss during gestation; however, neither the rate nor timing of pregnancy failure has been described in...
UNLABELLED
Previous reports indicate that red pandas () may experience fetal loss during gestation; however, neither the rate nor timing of pregnancy failure has been described in this species. The objective of this study was to utilize ultrasound video and images collected between 2010 and 2020 at the Cincinnati Zoo and Botanical Garden to better characterize pregnancy loss and fetal development. Trans-abdominal ultrasound examinations were performed on six female red pandas over a 10-year period, resulting in 12 profiles. Pregnancy was diagnosed via ultrasound in 10 of 12 profiles, and 40.0% of pregnancies showed evidence of fetal loss prior to parturition. Pregnancy loss was classified into lost (2 of 10; 20.0%), in which no cubs were produced, or partial loss (2 of 10; 20.0%), in which two concepti were visualized via ultrasound, but only one cub was born. Fetal loss occurred between days 51 and 23 pre-partum. Fetal growth characteristics were documented, including skeletal ossification (occurring between days 32 and 27 pre-partum), crown-rump length, head length, cranial length, and fetal heart rate (173-206 b.p.m.). These findings provide novel insights into pregnancy loss, may serve as a reference for milestones of fetal development, and may be useful in diagnosing pregnancy and assessing pregnancy loss in red pandas.
LAY SUMMARY
For many wildlife species, there is no non-invasive method of determining pregnancy; therefore, the rate of pregnancy loss oftentimes is unknown. Many red pandas in human care that are paired for breeding are observed exhibiting normal mating behaviors; however, only a relatively low proportion of females produce cubs. We utilized animals conditioned for ultrasound examination to diagnose pregnancy and characterize the incidence and timing of pregnancy loss. In total, 12 potential pregnancies were monitored, beginning after breeding season and ending ~2 weeks prior to anticipated cubbing. Of these, ten were (83.3%) were diagnosed as pregnant, with 40% undergoing either full or partial pregnancy loss. Fetal growth characteristics, such as body length and head size, are described which may be useful for monitoring pregnancies and estimating fetal age. Results of this study provide novel data on pregnancy loss in red pandas. Insights into the rate and timing of reproductive failure may illuminate causes and contributing factors, ultimately allowing for improvements in husbandry which may result in greater reproductive success of individuals recommended for breeding.
Topics: Abortion, Spontaneous; Ailuridae; Animals; Crown-Rump Length; Female; Gestational Age; Humans; Incidence; Pregnancy
PubMed: 35118406
DOI: 10.1530/RAF-21-0079 -
Scientific Reports Jan 2022Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic...
Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic vibration instead (Easy-ET method). The transferred embryos developed normally. Conventionally, stimulation is performed 7 × 30 s with 5 min intervals at the day before ET. However, this protocol is time-consuming because it imitates natural mating behavior. Here, we investigated pseudopregnancy induction with shorter stimulation times. Stimulation was performed 2 × 30 s, with 30 s intervals at the proestrus stage at the day before ET. Of the transferred pronuclear or two-cell embryos, 43% or 62% developed normally, respectively. Furthermore, 67% or 68% of transferred pronuclear or two-cell embryos in rats at estrus stage stimulated on the day of ET developed normally, respectively. Pseudopregnancy was successfully induced with shorter stimulation. Furthermore, this protocol may be used to perform a single-day stimulation and ET operation at the estrus stage.
Topics: Animals; Embryo Transfer; Female; Male; Pregnancy; Pseudopregnancy; Rats; Sound
PubMed: 35075219
DOI: 10.1038/s41598-022-05293-w -
Open Veterinary Journal 2021Fibroadenomatous changes (FAC) of the mammary gland in cats represents a benign, progesterone-associated fibroglandular proliferation of one or more mammary glands that...
BACKGROUND
Fibroadenomatous changes (FAC) of the mammary gland in cats represents a benign, progesterone-associated fibroglandular proliferation of one or more mammary glands that may occur in intact female cats at the time of puberty, during pregnancy or pseudopregnancy, or in female and male cats of any age under progestin treatment. Nowadays, the elective treatment of FAC is based on the progesterone antagonist aglepristone. This study aimed to report the treatment of FAC with a combination of drugs designed to preserve mammary gland integrity, even in pregnant cats.
CASE DESCRIPTION
Eight sexually intact female cats with FAC showed mammary glands that were symmetrically enlarged and inflamed in all cases, and the skin was ulcerated in six cats. Four cats were on days 25-32 of pregnancy at presentation. Non-pregnant cats were treated with aglepristone and with a dietary supplement containing maltodextrin and bromelain. The mammary glands were massaged daily with an emollient gel. If the gland was inflamed or ulcerated, broad-spectrum antimicrobial, and anti-inflammatory treatments were given, and the ulcers were treated topically with a hypericum and neem-based cream. Two of the four pregnant cats were treated with the same therapeutic schedule plus cloprostenol to facilitate uterine emptying. Two pregnant cats underwent the same protocol except for aglepristone, which was not administered to safeguard the litter and see if the adjuvant therapy worked independently. At term, they delivered four and three kittens, respectively, that were normally nursed and weaned after 40 days. In all the studied cases, the mammary gland reduced in size 2-3 weeks after the start of the treatment and completely remitted after 4-5 weeks.
CONCLUSION
This case series encourages adjuvant therapy in the course of FAC to preserve mammary gland integrity and functionality. Exploiting the natural decline of progesterone at the end of pregnancy (or pseudopregnancy), the therapy may also be used without aglepristone, when its use has other limitations.
Topics: Animals; Cats; Conservative Treatment; Female; Male; Pregnancy; Progesterone; Uterus
PubMed: 35070864
DOI: 10.5455/OVJ.2021.v11.i4.19 -
Reproduction in Domestic Animals =... Jan 2022The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant...
The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17β-oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non-pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non-pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post-ovulation, n = 5/group) and at different stages of pregnancy (n = 4-6/group): pre-implantation (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid-pregnancy to prepartum luteolysis. The main findings during non-pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.
Topics: Animals; Corpus Luteum; Diestrus; Dogs; Embryo Implantation; Estrogens; Female; Luteolysis; Pregnancy
PubMed: 34704613
DOI: 10.1111/rda.14032 -
Oxidative Medicine and Cellular... 2021The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of...
The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF during luteolysis, we hypothesized that the HIF-1 pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1 and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1 by echinomycin (Ech), a potent HIF-1 inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1 can also attenuate PGF -induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF treatment. Taken together, our results indicated that HIF-1 signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1 could obviously hamper the apoptosis of luteal cells and the process of luteal regression.
Topics: Animals; Corpus Luteum; Female; Hypoxia-Inducible Factor 1, alpha Subunit; Luteolysis; Pregnancy; Pseudopregnancy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species
PubMed: 34512862
DOI: 10.1155/2021/1764929