The growth factors amphiregulin and epiregulin are novel stimulators of feline ovarian granulosa cell functions.

This study aimed to investigate the impact of the epidermal growth factor receptor ligands amphiregulin (AREG) and epiregulin (EREG) on the fundamental functions of feline ovarian granulosa cells. Granulosa cells isolated from feline ovaries were incubated with AREG and EREG (0, 0.1, 1 or 10 ng/mL). The effects of these growth factors on cell viability, proliferation (assessed through BrdU incorporation), nuclear apoptosis (evaluated through nuclear DNA fragmentation) and the release of progesterone and estradiol were determined using Cell Counting Kit-8 assays, BrdU analysis, TUNEL assays and ELISAs, respectively. Both AREG and EREG increased cell viability, proliferation and steroid hormone release and reduced apoptosis. The present findings suggest that these epidermal growth factor receptor ligands may serve as physiological stimulators of feline ovarian cell functions.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

The in vitro mechanism of Vaspinregulates the proliferation and steroidogenesis of rat lutein cells.

OBJECTIVE: Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism. METHODS: Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway. RESULTS: Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels. CONCLUSION: Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.

Melatonin administration during the first half of pregnancy improves physiological response and reproductive performance of rabbits under heat stress conditions.

Context Melatonin may have a heat-stress-alleviating role during pregnancy. Aims To investigate the effects of melatonin administration during the first half of pregnancy on heat-tolerance capacity and pregnancy outputs of naturally heat-stressed rabbits. Methods Forty female rabbits were stratified equally into two experimental groups and daily received 1mg melatonin/kg body weight or not (control) for 15 consecutive days post-insemination. Heat tolerance indices, hormone profile, ovarian structures, and fetal loss were determined. Key results Treatment with melatonin significantly decreased respiration rate and rectal temperature, improved concentrations of nitric oxide, and tended to decrease malondialdehyde concentrations (P =0.064) compared to control. Melatonin treatment significantly increased concentrations of high-density lipoprotein, oestradiol, and progesterone compared to control. No significant differences in the numbers of visible ovarian follicles, corpora lutea, and total implantation sites on day 18 of pregnancy were observed between experimental groups. However, melatonin treatment significantly reduced the number of absorbed implantation sites and significantly improved amniotic fluid volume and conception rate compared to control. Conclusions Melatonin administration during the first half of pregnancy can improve reproductive performance of heat-stressed female rabbits. Implications Melatonin can improve fetal survivability via improving heat-tolerance capacity of does and steroidogenesis.

Timed-artificial insemination protocols for Bos indicus beef heifers: Evaluation of protocol length and variations in prostaglandin F2α treatments.

The aim was to compare reproductive outcomes of Nelore heifers submitted to timed AI (TAI) protocols, with 7 or 9 d of permanence of the intravaginal progesterone (P4) device and different times of prostaglandin F2α (PGF) administration, for first (n = 935) and second (n = 530) services. On Day -24, heifers without corpus luteum (CL) underwent a protocol for induction of ovulation. On Day 0, heifers received a P4 device (0.5 g) and 1.5 mg estradiol (E2) benzoate. In order for the TAI to be carried out on the same day, these treatments were performed 2 d later on the heifers treated with the 7-d protocol. Additionally, heifers received 0.5 mg PGF at different times, resulting in four experimental groups: 9dP4-PGFd9 (n = 365); 9dP4-PGFd7 (n = 369); 9dP4-PGFd0&9 (n = 364); 7dP4-PGFd0&7 (n = 367). These nomenclatures indicate for how many d the P4 device was kept and the specific day on which PGF was given. At P4 removal, all heifers received 0.5 mg E2 cypionate and 200 IU eCG, and TAI was performed 2 d later. Effects were considered significant when P ≤ 0.05 (superscript letters a,b) whereas a tendency was assumed when 0.05 < P ≤ 0.10. Groups 9dP4-PGFd0&9 and 7dP4-PGFd0&7 had lower percentage of heifers with CL at P4 removal. The diameter (mm) of the dominant follicle (DF) was affected by treatment at P4 removal (9dP4-PGFd9: 11.3 ± 0.3b; 9dP4-PGFd7: 11.8 ± 0.2ab; 9dP4-PGFd0&9: 12.6 ± 0.2a; 7dP4-PGFd0&7: 10.8 ± 0.2c) and at TAI (9dP4-PGFd9: 12.7 ± 0.3ab; 9dP4-PGFd7: 13.2 ± 0.2a; 9dP4-PGFd0&9: 13.4 ± 0.2a; 7dP4-PGFd0&7: 12.4 ± 0.3b). Expression of estrus (%) was affected by treatment (9dP4-PGFd9: 89.6a; 9dP4-PGFd7: 93.5a; 9dP4-PGFd0&9: 88.2ab; 7dP4-PGFd0&7: 85.6b). There were no differences among treatments for P/AI on Day 40 (30-35 d post AI), final P/AI (between Day 70 and parturition) and pregnancy loss (between Day 40 and final P/AI). When the permanence of the P4 device was compared, regardless of PGF treatments, 9-d protocols resulted in greater DF diameter at P4 removal and at TAI, and greater expression of estrus (90.4 vs. 85.6%) than the 7-d protocol. Despite that, the 7-d protocol resulted in greater P/AI on Day 40 (55.3 vs. 49.1%). In addition, there was an interaction between protocol duration and body weight, in which heavier heifers (≥ 307 kg) had greater P/AI when treated with the 7-d protocol, in comparison to 9-d. In conclusion, longer TAI protocols (9 d of P4 device duration) resulted in greater DF diameter and expression of estrus. However, the shorter TAI protocol (7 d of P4 device duration) produced greater P/AI on Day 40, particularly in heavier heifers. Within 9-d protocols, the additional dose of PGF on Day 0 or the anticipation of the PGF to Day 7 did not influence fertility.

Biochemical profile and oocyte quality of primiparous Bos indicus cows submitted to a timed artificial insemination protocol.

The objective of this study was to verify the causes of the lower response of primiparous Bos indicus cows to the ovulation synchronization protocol. Two experiments were performed to evaluate the biochemical profile, oocyte and follicular cell quality (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, suckled primiparous (n = 24) and multiparous cows (n = 24) were submitted to ovum pick up (OPU). On Day 0 (D0), all cows received 2 mg of estradiol benzoate (EB) and an intravaginal progesterone insert (P4). Five days (D5) after the first hormonal administration (EB + P4), all follicles larger than 3 mm were counted on each ovary, and ovum pick-up (OPU) was performed. On day 12 (D12), the intravaginal progesterone insert was removed, and measurement and aspiration of the largest follicle were performed. Blood samples were collected on D5 and D12 to evaluate the concentrations of glucose, cholesterol, NEFA, IGF-1 and insulin. In Experiment 2, suckled primiparous (n = 50) and multiparous (n = 50) cows were subjected to an ovulation synchronization protocol based on E2/P4 (D0: 2 mg EB plus P4 intravaginal insert; D8: 500 µg of cloprostenol, 1 mg cypionate estradiol and 300UI of eCG; D10: artificial insemination). In addition, blood samples were collected on D10 for evaluation of the same hormones and metabolites described in Experiment 1. In all studies, calves remained with the cows during the experimental period. In experiment 1, the number of oocytes grade 1 (P = 0.83), grade 2 (P = 0.23) and grade 3 (P = 0.51), total number of retrieved oocytes (P = 0.14), oocyte quality index (P = 0.93) and total viable oocytes (P = 0.38) did not differ between primiparous and multiparous cows. The number of follicles at the time of follicular aspiration (20.7 ± 1.5 vs. 18.0 ± 1.9; P = 0.05) and the diameter of the largest follicle on D12 (13.5 ± 0.6 vs. 11.4 ± 0.6; P = 0.04) were greater in multiparous cows, and the number of degenerated oocytes was greater in primiparous cows (1.9 ± 0.7 vs. 1.2 ± 0.3; P = 0.05). On D5, the concentrations of IGF-1 (P = 0.47), insulin (P = 0.08), total cholesterol (P = 0.47), NEFA (P = 0.77) and glucose (P = 0.55) in the blood and IGF-1 (P = 0.97) and insulin (P = 0.11) in the follicular fluid did not differ between parity groups. On D12, there was no difference in the concentrations of IGF-1 (P = 0.26), total cholesterol (P = 0.32), NEFAs (P = 0.31) and glucose (P = 0.93) in the blood between primiparous and multiparous cows, however, the serum insulin concentration (P = 0.04) was higher in primiparous cows. There was no correlation between serum and follicular fluid insulin concentrations (r = 0.17; P = 0.31), however, there was a low correlation between serum and follicular fluid IGF-1 concentrations (r = 0.47; P = 0.002). Quantification of transcripts did not differ between parity groups. In experiment 2, concentrations of NEFA (P = 0.12) and insulin (P = 0.16) in the blood and P/AI (P = 0.93) did not differ between parity [60 % (30/50) primiparous vs. 60 % (30/50) multiparous]. In contrast, blood concentrations of IGF-1 (P = 0.0001), total cholesterol (P = 0.005) and glucose (P = 0.01) were greater in primiparous cows. It was concluded that the oocyte quality and expression of the genes evaluated in the granulosa cells were not different between primiparous and multiparous cows. Unexpectedly, the pregnancy rate did not differ between parity. Nevertheless, the blood concentrations of IGF-1, total cholesterol and glucose were greater in primiparous cows.

Optimizing ReBreed21 II: Fertility and reproductive efficiency in different parities during a shortened breeding season in beef cattle.

This study evaluated the efficiency of a rapid reinsemination program allowing timed AI (TAI) every 21d (ReBreed21) in a commercial beef cow-calf operation. Nelore females from different parities (n = 2085) were synchronized for first TAI (D0 = TAI) using an estradiol/progesterone (E2/P4) protocol and assigned to one of three reinsemination programs: Resynch33 (n = 753), traditional resynch program with second TAI at D42 after first TAI; ReBreed21 (n = 687); or ReBreed21+EC (n = 670). The ReBreed females (n = 1357) received intravaginal P4 insert on D12, on D19 P4 was removed, and a dose of equine chorionic gonadotropin (eCG) was administered, then, ReBreed21 females received 0.6 mg of EC (ReBreed21+EC) or nothing (ReBreed21) and on D21, nonpregnancy (NP) was determined using Doppler ultrasound to detect corpus luteum (CL) blood flow (BF) (NP: <25 % BF pixels of total CL area) and NP cows received immediate TAI and GnRH to induce ovulation. Pregnancy diagnosis was performed at D33 after TAI following all TAIs. Cows considered pregnant at D21, based on CL BF, but NP on D33 were designated False-Positives (FP) and false negatives (FN) were number of nonpregnant cows/heifers on d21 based on the CL BF found to subsequently be pregnant on D33 divided by the total number pregnant. Pregnancy/AI (P/AI) did not differ for the first TAI (55.1 %) among the treatments. Heifers had similar P/AI at the second AI in all groups and similar to the first AI. Primiparous had greater P/AI in ReBreed21+EC and Resynch33 at s TAI compared to ReBreed21, 51.7 %, 55.8 %, 34.2 %, respectively. Multiparous had greater P/AI at second TAI in Resynch33 (60.9 %) than ReBreed21 programs (34.7 %). The percentage FP and FN among ReBreed21 programs did not differ, 13.8 and 0.2 %, respectively. Overall accumulative pregnancies on D21 of the breeding season were greater for ReBreed21 and ReBreed21+EC than Resynch33 (69.7 %, 71.6 %, and 55.5 %, respectively). However, on D42 of the breeding season, only heifers had greater pregnancies in ReBreed21 programs than Resynch33 (73.3 %, 74.3.6 %, and 63.2 %, respectively). Average days to pregnancy were less (P = 0.01) for ReBreed21 and ReBreed21+EC than Resynch33. Thus, the ReBreed21 strategy can improve the efficiency of TAI programs in beef cattle. Of interest, ReBreed21 was particularly effective in nulliparous, somewhat effective in primiparous when EC was added to the program, but relatively ineffective in multiparous beef cattle.

Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Prenatal progesterone treatment modulates fetal brain transcriptome and impacts adult offspring behavior in mice.

Maternal exposure to elevated levels of steroid hormones during pregnancy is associated with the development of chronic conditions in offspring that manifest in adulthood. However, the effects of progesterone (P4) administration during early pregnancy on fetal development and subsequent offspring behavior remain poorly understood. In this study, we aimed to investigate the effects of P4 treatment during early pregnancy on the transcript abundance in the fetal brain and assess the behavioral consequences in the offspring during adolescence and adulthood. Using RNA-seq analysis, we examined the impact of P4 treatment on the fetal brain transcriptome in a dosage-dependent manner. Our results revealed differential regulation of genes involved in neurotransmitter transport, synaptic transmission, and transcriptional regulation. Specifically, we observed bidirectional regulation of transcription factors (TFs) by P4 at different doses, highlighting the critical role of these TFs in neurodevelopment. To assess behavioral outcomes, we conducted open field and elevated plus maze tests. Offspring treated with low-dose P4 (LP4) displayed increased exploratory behavior during both adolescence and adulthood. In contrast, the high-dose P4 (HP4) group exhibited impaired exploration and heightened anxiety-like behaviors compared to the control mice. Moreover, in a novel object recognition test, HP4-treated offspring demonstrated impaired object recognition memory during both developmental stages. Additionally, both LP4 and HP4 groups showed reduced social interaction in the three-chamber test. These results suggest that prenatal exposure to P4 exerts a notable influence on the expression of genes associated with neurodevelopment and may induce alterations in behavioral characteristics in progeny, highlighting the need to monitor progesterone levels during pregnancy for long-term impacts on fetal brain development and behavior.

Effects of short and long - term nutrition and progesterone supplementation on the success of fixed - time artificial insemination in the ewe.

The success of fixed - time artificial insemination (AI) in the ewe is variable due to poor synchrony of estrus. We examined the effects of long-term nutrition (LTN; low, medium, high - 6 months), short-term nutrition (STN; 1.0 M, 1.5 M - 14 days) and progesterone supplementation (P; single pessary, replacement on Day 9) on synchrony and reproductive outcomes. High LTN advanced (P < 0.05) estrus, increased (P = 0.06) pregnancy (range 71.1 - 81.1%) and improved (P < 0.01) litter size (range 1.30 - 1.50). STN increased (P < 0.05) pregnancy (79.0 versus 72.3%) but not litter size or timing of estrus. A LTN x STN interaction (P < 0.01) for time of estrus indicates that the effects of LTN were moderated by STN depending on the level of LTN. Pessary replacement delayed (P < 0.05) the onset of estrus, improved synchrony but did not affect pregnancy or litter size. High LTN increased (P < 0.05) the number of large (≥ 3.8 mm) and medium - size follicles (2.0 - 3.7 mm) but the diameter of large follicles tended to be reduced (P = 0.08) on Day 12. STN did not affect follicle number or size whilst P reduced (P < 0.05) the diameter of large follicles on Day 12 (4.83 versus 5.10 mm) and increased the number of medium - size follicles (3.56 versus 2.74 mm). In conclusion, both LTN and STN are major sources of variability in AI programs whilst pessary replacement has potential to reduce variability.

Survival and hormone production of isolated mouse follicles in three-dimensional artificial scaffolds after stimulation with bpV(HOpic).

PURPOSE: To preserve fertility before gonadotoxic therapy, ovarian tissue can be removed, cryopreserved, and transplanted back again after treatment. An alternative is the artificial ovary, in which the ovarian follicles are extracted from the tissue, which reduces the risk of reimplantation of potentially remaining malignant cells. The PTEN inhibitor bpV(HOpic) has been shown to activate human, bovine and alpacas ovarian follicles, and it is therefore considered a promising substance for developing the artificial ovary. The purpose of this study was to examine the impact of different scaffolds and the vanadate derivative bpV(HOpic) on mice follicle survival and hormone secretion over 10 days. METHODS: A comparative analysis was performed, studying the survival rates (SR) of isolated mice follicle in four different groups that differed either in the scaffold (polycaprolactone scaffold versus polyethylene terephthalate membrane) or in the medium-bpV(HOpic) versus control medium. The observation period of the follicles was 10 days. On days 2, 6, and 10, the viability and morphology of the follicles were checked using fluorescence or confocal microscopy. Furthermore, hormone levels of estrogen (pmol/L) and progesterone (nmol/L) were determined. RESULTS: When comparing the SR of follicles among the four groups, it was observed that on day 6, the study groups utilizing the polycaprolactone scaffold with bpV(HOpic) in the medium (SR: 0.48 ± 0.18; p = 0.004) or functionalized in the scaffold (SR: 0.50 ± 0.20; p = 0.003) exhibited significantly higher survival rates compared to the group using only the polyethylene terephthalate membrane (SR: 0). On day 10, a significantly higher survival rate was only noted when comparing the polycaprolactone scaffold with bpV(HOpic) in the medium to the polyethylene terephthalate membrane group (SR: 0.38 ± 0.20 versus 0; p = 0.007). Higher levels of progesterone were only significantly associated with better survival rates in the group with the polycaprolactone scaffold functionalized with bpV(HOpic) (p = 0.017). CONCLUSION: This study demonstrates that three-dimensional polycaprolactone scaffolds improve the survival rates of isolated mice follicles in comparison with a conventional polyethylene terephthalate membrane. The survival rates slightly improve with added bpV(HOpic). Furthermore, higher rates of progesterone were also partly associated with improved survival.

Optimizing ReBreed21 I: Evaluation of endocrine and ovarian dynamics in non-bred Bos indicus heifers.

The present study evaluated follicular and endocrine dynamics during ReBreed21, a reproductive strategy that allows resynchronization of ovulation every 21 days in Bos indicus (Nelore) heifers. A synchronized estrous cycle was induced using a standard timed ovulation protocol (d -10: P4 implant inserted + 2 mg estradiol benzoate; d -2: P4 removed+ 0.5 mg cloprostenol + 0.6 mg estradiol cypionate + 200 IU equine chorionic gonadotropin (eCG); d0: 8.4 µg buserelin) without AI to ensure nonpregnancy in heifers. Day of GnRH was designated d0 of estrous cycle. On d12, heifers (n = 80) were randomized into three experimental groups: (1) ReBreed21 (n = 28) d12 P4 device inserted, d19 P4 device withdrawal plus 200 IU eCG, and d21 8.4 µg buserelin (GnRH); (2) ReBreed21+G (n = 26) same as ReBreed21 plus GnRH (16.8 µg) treatment on d12; and (3) Control (n = 26) no treatment. ReBreed21+G increased two-fold (62.9%; 18/26) percentage of heifers with synchronized follicular wave emergence compared to Control (34.6%; 9/26) whereas ReBreed21 (53.6%; 15/28) was intermediate. The ReBreeed21 groups (eCG on d19) increased (P < 0.01) follicular growth between d19 and d21 in ReBreed21 (2.3 ± 0.2 mm) and ReBreed21+G (3.4 ± 0.2 mm) compared with Control (1.2 ± 0.3 mm), resulting in greater (P < 0.01) follicle diameter on d21 for ReBreed21 (10.7 ± 0.4 mm) and ReBreed21+G (10.8 ± 0.4 mm) compared with Control (9.1 ± 0.5 mm). Structural luteolysis was similar among groups (P = 0.51), although the average day when P4 was <1 ng/mL was later (P < 0.01) for ReBreed21 (20.5 ± 0.2) and ReBreed21+G (20.7 ± 0.2) compared to Control (19.2 ± 0.4). Overall ovulation at the end of the estrous cycle was increased (P = 0.03) for ReBreed21 groups (83.3%; 45/54) compared with Control (57.7%; 15/26). Synchronized ovulation on day 22-23 was greater (P < 0.01) for ReBreed21 (78.6%; 22/28) and ReBreed21+G (76.9%; 20/26) compared with Control (30.8%; 8/26). Thus, the ReBreed21 resynchronization program produced acceptable endocrine and follicular dynamics, including synchronized ovulation at the end of the protocol in nonpregnant heifers providing good rationale for testing the fertility and practical implementation of this protocol under field conditions.

Estrogens improve the pregnancy rate in cattle: A review and meta-analysis.

Estrogens have proven to be effective in bovine estrus induction protocols. Considering the extensive use of these products in large-scale estrus synchronization, the primary objective of the present study was to assess their effects on pregnancy rate (PR) using a meta-analysis approach. A total of 797 papers were screened from three major databases (PubMed, Web of Science, Scopus). Sixty-one studies were eligible for inclusion in the meta-analysis. The pregnancy status (success or failure) at 30 days post-insemination was considered as the effect size data. The odds ratios (OR) of PR were evaluated by considering the effects of estrogens in groups with or without estrogen intervention. The impact of estrogen (including factors such as type, dose, and time of administration) and animal characteristics (such as breed, type, and parity) was taken into account when assessing the effectiveness of estrogen response as PR. The results showed an OR of 1.25 (95% CI: 1.15-1.36; P = 0.000) for PR in animals that received estrogen compared to cattle that did not receive estrogen. Estradiol benzoate (OR = 1.3) and estradiol cypionate (OR = 1.2), with doses ranging from 1 to 3 mg (OR = 1.13-1.7), significantly increased the OR of PR. In terms of PR, beef cattle exhibited a higher odds ratio (OR = 1.4; P = 0.000) compared to dairy cattle (OR = 1.1; P = 0.09). The administration of estrogens in the estrus synchronization protocol significantly improved PR in both artificial insemination (OR = 1.2; P = 0.000) and embryo transfer (OR = 1.3; P = 0.033) programs. In summary, incorporating estrogens into estrus induction protocols led to an enhancement of the OR of PR among cattle.

Ulipristal (UPA) effects on rat ovaries: Unraveling follicle dynamics, ovulation inhibition, and safety implications for prolonged use.

Ulipristal (UPA), a selective progesterone receptor modulator, has both agonistic and antagonistic effects on progesterone receptors. UPA suppresses ovulation by inhibiting the luteinizing hormone (LH) surge from the pituitary gland; however, the direct effect of UPA on ovarian tissue remains poorly studied. In the present study, we examined the effects of UPA on the ovaries of rats. Rats were treated for 28 days with UPA, and the effects of UPA on ovarian tissue were examined histologically and the expression of antioxidant genes and cell death markers were also investigated. UPA treatment increased the number of primordial follicles at each treatment group, primordial follicles increased at all dose levels, but the size/magnitude of the effect decreased with the increasing dose. The number of primary and antral follicles tended to increase with increasing UPA levels. Furthermore, the decrease in primary follicle number could be attributed to the exhaustion of follicles, but the examination of proliferation markers, oxidative stress markers, and cell death markers revealed no remarkable toxic effects on ovarian tissues. These results suggest that UPA treatment promotes follicle development at each stage but inhibits ovulation by suppressing the LH surge, resulting in an increase in atretic follicles or unruptured luteinized cysts. These results suggest that UPA may not have both toxic effects on the ovary and a direct local effect on ovarian follicles, but we should be careful about the effects of prolonged UPA treatment in patients with uterine fibroids on their future fecundity.

Effect of estrogen and progesterone on intracellular free zinc and zinc transporter expression in bovine oviduct epithelial cells.

Zinc (Zn) plays essential roles in numerous cellular processes. However, there is limited understanding of Zn homeostasis within the bovine reproductive system. This study investigated the influence of estradiol (E2) and progesterone (P4) on Zn transporter expression and intracellular free Zn levels in bovine oviduct epithelial cells (BOEC). For this purpose, cells were harvested from slaughtered cows and cultured in vitro. Intracellular Zn concentrations were measured using FluoZin-3AM staining, while real-time polymerase chain reaction assessed Zn transporter gene expression and quantification. Overall, our results confirmed the gene expression of all the evaluated Zn transporters (ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9), denoted and the active role of E2 and P4 in intracellular Zn regulation. Our findings suggest an interaction between Zn, E2 and P4.

Progesterone induces neuroprotection associated with immune/inflammatory modulation in experimental traumatic brain injury.

An imbalance of immune/inflammatory reactions aggravates secondary brain injury after traumatic brain injury (TBI) and can deteriorate clinical prognosis. So far, not enough therapeutic avenues have been found to prevent such an imbalance in the clinical setting. Progesterone has been shown to regulate immune/inflammatory reactions in many diseases and conveys a potential protective role in TBI. This study was designed to investigate the neuroprotective effects of progesterone associated with immune/inflammatory modulation in experimental TBI. A TBI model in adult male C57BL/6J mice was created using a controlled contusion instrument. After injury, the mice received consecutive progesterone therapy (8 mg/kg per day, i.p.) until euthanized. Neurological deficits were assessed via Morris water maze test. Brain edema was measured via the dry-wet weight method. Immunohistochemical staining and flow cytometry were used to examine the numbers of immune/inflammatory cells, including IBA-1 + microglia, myeloperoxidase + neutrophils, and regulatory T cells (Tregs). ELISA was used to detect the concentrations of IL-1ß, TNF-α, IL-10, and TGF-ß. Our data showed that progesterone therapy significantly improved neurological deficits and brain edema in experimental TBI, remarkably increased regulatory T cell numbers in the spleen, and dramatically reduced the activation and infiltration of inflammatory cells (microglia and neutrophils) in injured brain tissue. In addition, progesterone therapy decreased the expression of the pro-inflammatory cytokines IL-1ß and TNF-α but increased the expression of the anti-inflammatory cytokine IL-10 after TBI. These findings suggest that progesterone administration could be used to regulate immune/inflammatory reactions and improve outcomes in TBI.

Magnitude and persistence of higher estrus-associated temperatures in beef heifers and suckled cows.

Higher estrus-associated temperatures (HEAT) are a hallmark feature in sexually active females. The overarching aim of this study was to characterize the variability, magnitude, and persistence of HEAT in heifers and suckled beef cows as well as identify associated factors when occurring during thermoneutral conditions at the onset of the spring breeding season. In both heifers and cows, estrus was induced using a 7-d controlled internal drug release (CIDR)-PGF2α protocol. Vaginal temperature after prostaglandin F2α administration was recorded every 5 min using a Thermochron iButton affixed to a blank CIDR (containing no progesterone). Estrus was defined as when a heifer first stood to be mounted or when a cow had an Estrotect patch score of 3 or 4. Level of HEAT varied among individual animals. When comparing common HEAT variables using a mixed model with date nested within a year, maximum HEAT (39.9 ±â€…0.1 and 40.0 ±â€…0.1 °C) and duration (15.5 ±â€…0.8 and 15.4 ±â€…0.7) were similar in heifers and cows, respectively. However, the magnitude and persistence of HEAT differed. Total area under the HEAT curve was 117.1 ±â€…13.5 and 158.7 ±â€…12.3 for heifers vs cows, respectively (P = 0.0571). Further, 42.9% of heifers and 49% of cows had maximum HEAT ≥ 40 °C which persisted up to 6.5 and 10 h, respectively. When ambient conditions were predominantly thermoneutral, temperature humidity index had minimal impact on HEAT (mixed model, repeated measures over time). Toward identifying associated factors with different aspects of HEAT using best fit hierarchical linear regression models, baseline vaginal temperature and baseline duration were the most highly associated independent variables. Follicle size, estradiol and progesterone levels, and other available animal-related variables (e.g., age, weight, hair coat score) explained only a small amount of variation in HEAT. In summary, level of HEAT varies in estrus females even under thermoneutral conditions. Because HEAT can persist for an extended time, direct effects on fertility important components are unavoidable. Whether HEAT is a good or bad component of the periovulatory microenvironment is the basis of ongoing and future studies.

When striving for a pregnancy, estrus is a critically important event. Higher estrus-associated temperatures (HEAT) are a hallmark feature in sexually active females. The importance of HEAT for pregnancy, however, remains unclear. Toward filling this critical knowledge gap, efforts described in the current study focused on examining variability of HEAT in individual animals, 2) defining the magnitude and persistence of HEAT, 3) identifying HEAT-associated factors, and 4) examining the similarity of HEAT between heifers and suckled beef cows when occurring at the onset of a spring breeding season. Although the magnitude and persistence of HEAT varied, 42.9% of heifers and 49% of cows reached temperatures ≥ 40 °C which in some cases persisted up to 6.5 and 10 h, respectively. When attempting to identify factors that could explain why some females exhibiting estrus remained hot for an extended time, available animal and environmental data contributed little. Even so, because HEAT can persist for an extended time, direct effects on fertility important components are unavoidable. Whether too much HEAT is good or bad for pregnancy is the basis of ongoing and future studies.

Photoperiod, but not progesterone, has a strong impact upon the transcriptome of the medio-basal hypothalamus in female goats and ewes.

Photoperiod is the main environmental driver of seasonal responses in organisms living at temperate and polar latitudes. Other external cues such as food and temperature, and internal cues including hormones, intervene to fine-tune phasing of physiological functions to the solar year. In mammals, the medio-basal hypothalamus (MBH) is the key integrator of these cues, which orchestrates a wide array of seasonal functions, including breeding. Here, using RNAseq and RT-qPCR, we demonstrate that molecular components of the photoperiodic response previously identified in ewes are broadly conserved in does (female goats, Capra hircus), with a common core of ∼50 genes. This core group can be defined as the "MBH seasonal trancriptome", which includes key players of the pars tuberalis-tanycytes neuroendocrine retrograde pathway that governs intra-MBH photoperiodic switches of triiodothyronine (T3) production (Tshb, Eya3, Dio2 and SlcO1c1), the two histone methyltransferases Suv39H2 and Ezh2 and the secreted protein Vmo1. Prior data in ewes revealed that T3 and estradiol (E2), both key hormones for the proper timing of seasonal breeding, differentially impact the MBH seasonal transcriptome, and identified cellular and molecular targets through which these hormones might act. In contrast, information regarding the potential impact of progesterone (P4) upon the MBH transcriptome was nonexistent. Here, we demonstrate that P4 has no discernible transcriptional impact in either does or ewes. Taken together, our data show that does and ewes possess a common core set of photoperiod-responsive genes in the MBH and conclusively demonstrate that P4 is not a key regulator of the MBH transcriptome.

Progesterone-mediated remodeling of the maternal-fetal interface by a PGRMC1-dependent mechanism.

Implantation and maintenance of pregnancy involve intricate immunological processes that enable the developing fetus to coexist with the maternal immune system. Progesterone, a critical hormone during pregnancy, is known to promote immune tolerance and prevent preterm labor. However, the mechanism by which progesterone mediates these effects remains unclear. In this study, we investigated the role of the non-classical progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling at the maternal-fetal interface. Using JEG3 cells, a trophoblast model cell line, we observed that progesterone stimulation increased the expression of human leukocyte antigen-C (HLA-C) and HLA-G, key molecules involved in immune tolerance. We also found that progesterone upregulated the expression of the transcription factor ELF3, which is known to regulate trophoblast-specific HLA-C expression. Interestingly, JEG3 cells lacked expression of classical progesterone receptors (PRs) but exhibited high expression of PGRMC1, a finding we confirmed in primary trophoblasts by mining sc-RNA seq data from human placenta. To investigate the role of PGRMC1 in progesterone signaling, we used CRISPR/Cas9 technology to knockout PGRMC1 in JEG3 cells. PGRMC1-deficient cells showed a diminished response to progesterone stimulation. Furthermore, we found that the progesterone antagonist RU486 inhibited ELF3 expression in a PGRMC1-dependent manner, suggesting that RU486 acts as a progesterone antagonist by competing for receptor binding. Additionally, we found that RU486 inhibited cell invasion, an important process for successful pregnancy, and this inhibitory effect was dependent on PGRMC1. Our findings highlight the crucial role of PGRMC1 in mediating the immunoregulatory effects of progesterone at the maternal-fetal interface.

Local effect of allopregnanolone in rat ovarian steroidogenesis, follicular and corpora lutea development.

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3ß-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABAA receptor increased after ALLO treatment. To evaluate if the ovarian GABAA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABAA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.