Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Interferon-tau infusion into the ovine corpus luteum delays luteolysis.

Conceptus-derived interferon-tau (IFNT) initiates maternal recognition of pregnancy in ewes by paracrine actions on the endometrium and endocrine action on the corpus luteum (CL). To examine the effect of IFNT on the CL without inducing IFN stimulated genes (ISGs) in the endometrium, recombinant ovine IFNT (roIFNT) or bovine serum albumin (BSA) was delivered directly into CLs via osmotic pumps at a rate of 10, 50 or 100 ng/h from days 9 to 12 of the estrous cycle. Endometrial and CL samples were collected on day 12. Fifty ng/h of roIFNT induced ISG15 in the CL on day 12 without affecting endometrial ISG15 concentrations. In a second experiment, roIFNT (50 ng/h) was infused into the CL from days 10 to 17 of the estrous cycle and serum samples were collected daily. Serum progesterone concentrations were significantly higher on days 15 to 17 in roIFNT-infused ewes compared to controls. Levels of LHCGR, STAR, CYP11A1, HSL, OPA1 and PKA mRNA and proteins were higher in the roIFNT-infused CLs compared to the controls. Levels of ISG15 and MX1 mRNA increased in the CLs of roIFNT-infused ewes but not in the endometrium. Endometrial ESR1 mRNA and protein concentrations were higher in the controls compared to roIFNT-infused ewes. In conclusion, intra-luteal delivery of roIFNT induced ISGs, stabilized steroidogenesis in the CL and delayed luteolysis without inducing endometrial ISGs. Inhibition of ESR1 in the endometrium of roIFNT-infused ewes was observed suggesting that direct delivery of IFNT to the CL has an additional anti-luteolytic effect on the endometrium.

Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

Investigation of luteal HCG supplementation in GnRH-agonist-triggered fresh embryo transfer cycles: a randomized controlled trial.

RESEARCH QUESTION: Does splitting the human chorionic gonadotrophin (HCG) support in IVF cycles triggered by a gonadotrophin-releasing hormone agonist result in a better progesterone profile? DESIGN: Randomized controlled three-arm study, performed at the Fertility Clinic, Odense University Hospital, Denmark. Patients with 12-25 follicles ≥12 mm were randomized into three groups: Group 1 - ovulation triggered with 6500 IU HCG; Group 2 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1500 IU HCG on the day of oocyte retrieval (OCR); and Group 3 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1000 IU HCG on the day of OCR and 500 IU HCG on OCR + 5. All groups received 180 mg vaginal progesterone. Progesterone concentrations were analysed in eight blood samples from each patient. RESULTS: Sixty-nine patients completed the study. Baseline and laboratory data were comparable. Progesterone concentration peaked on OCR + 4 in Groups 1 and 2, and peaked on OCR + 6 in Group 3. On OCR + 6, the progesterone concentration in Group 2 was significantly lower compared with Groups 1 and 3 (P = 0.003 and P < 0.001, respectively). On OCR + 8, the progesterone concentration in Group 3 was significantly higher compared with the other groups (both P<0.001). Progesterone concentrations were significantly higher in Group 3 from OCR + 6 until OCR + 14 compared with the other groups (all P ≤ 0.003). Four patients developed ovarian hyperstimulation syndrome in Group 3. CONCLUSION: Sequential HCG support after a GnRH agonist trigger provides a better progesterone concentration in the luteal phase.

Symptomatic corpus luteum hemorrhage in adolescent females with ITP.

Patients with immune thrombocytopenia (ITP) usually present with minor mucocutaneous bleeding. Corpus luteum hemorrhage (CLH) is generally asymptomatic but may, rarely, lead to severe intraperitoneal bleeding, mostly in patients with coagulation disorders. CLH causing intraperitoneal bleeding has only been described in few individuals with ITP. The objective of this retrospective observational study was to assess the clinical course and incidence of symptomatic CLH in adolescent females with newly diagnosed or chronic ITP. Additionally, a comprehensive literature review was conducted to scrutinize cases of pediatric female patients with ITP, complicated by CLH. We identified three patients with ITP and hemoperitoneum secondary to CLH. They presented with acute abdominal pain, had severe thrombocytopenia (platelet counts below 20 × 109/L), and required blood transfusions as well as ITP-directed therapy. All the patients were hemodynamically stable and did not require emergency surgical intervention.  Conclusion: CLH could potentially pose a significant complication in the context of adolescent females with ITP, requiring a strong index of suspicion to direct expedient therapy. What is Known: • Immune thrombocytopenia is typically associated with minor bleeding tendency. • Corpus luteum hemorrhage is generally asymptomatic; however, in women with bleeding disorders, it has the potential to result in substantial intra-abdominal bleeding. What is New: • Corpus luteum hemorrhage leading to intra-abdominal bleeding is a potential severe complication of immune thrombocytopenia in adolescent females.

Lipid droplets synthesized during luteinization are degraded after pregnancy.

After pregnancy, the corpus luteum (CL) functions as a transient endocrine gland that produces progesterone, which is necessary to maintain pregnancy. To maintain constant progesterone production, CLs are enriched in lipids as its precursors. Lipid droplets (LDs) are organelles that originate from the endoplasmic reticulum and store neutral lipids such as triacylglycerols and cholesteryl esters. The size and number of LDs in a cell are regulated by LD-associated proteins that coat their surface. LD degradation is regulated by either neutral lipid hydrolases (lipolysis), selective autophagic mechanism (lipophagy), or both. Mammalian CLs are long known to be enriched in LDs, but LDs are rapidly depleted after pregnancy and reappear near the time of delivery. In this present study, we hypothesized that LDs synthesized by luteinization are massively degraded after pregnancy. Using mCherry-HPos mice, in which LD synthesis can be visualized in vivo, we found that LD synthesis, which was activated during luteal development, was suppressed after implantation. In CLs, LD synthesis remained low during pregnancy, but was reactivated before and after delivery. These changes in LDs were confirmed using electron microscopy and immunostaining. Furthermore, LD degradation was mediated by lipolysis rather than lipophagy. In summary, our findings indicate that luteinization-induced LD synthesis is suppressed after pregnancy onset and that CLs are lipid-poor during pregnancy because LDs stored during luteal development are extensively degraded by lipolysis.

Role of chemokines in regulating luteal and uterine functions in pregnant cows.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

Autophagy in the corpus luteum correlates with tissue growth in pregnant rats.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

The fertility of dairy heifers and cows is not influenced by the follicular wave of the ovulatory follicle.

Two studies were conducted to evaluate the effects of the follicular wave on ovarian function and fertility in dairy heifers and lactating cows. In study 1, the estrous cycle of the selected Holstein heifers was initially synchronized using two intra-muscular prostaglandin F2α (PGF2α) administrations 11 days apart. Heifers in group FFW (n = 14) received an intra-muscular 500 µg PGF2α administration on day 7 after detecting standing estrus, while Heifers in group SFW (n = 14) were administered PGF2α 13 days after detecting standing estrus. The pregnancy rates of FFW (n = 98) and SFW (n = 100) heifers were also determined 35-37 days after artificial insemination (AI). In Study 2, healthy Holstein lactating cows (n = 28) were randomly assigned to either the FFW (n = 14) or SFW (n = 14) groups. The estrous cycles of the cows were presynchronized using two intra-muscular administrations of PGF2α given 14 days apart. Then, the emergences of the follicular waves were induced using an Ovsynch protocol. The pregnancy rate of FFW (n = 99) versus SFW (n = 98) cows was also determined 35-37 days after AI. The ovulatory follicle and corpus luteum (CL) resulting from the ovulatory follicle of FFW were larger than those of the dominant follicle and the CL of SFW in dairy heifers and lactating cows. However, the pregnancy rate did not differ between the FFW and SFW groups in heifers and lactating cows 35-37 days after AI. In conclusion, although the characteristics of the ovulatory follicles in FFW versus SFW animals differed, the follicular wave in dairy heifers or lactating cows did not affect fertility.

Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have other causes than pregnancy losses. We enrolled Holstein dairy cows ≥50 d in milk (DIM) from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an artificial insemination (AI) or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a CL on d17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, body condition score, and the presence of a corpus luteum (CL) at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92, 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.

Pathologic maternal and neonatal outcomes associated with programmed embryo transfer: potential etiologies and strategies for prevention.

PURPOSE: In the first of two companion papers, we comprehensively reviewed the recent evidence in the primary literature, which addressed the increased prevalence of hypertensive disorders of pregnancy, late-onset or term preeclampsia, fetal overgrowth, postterm birth, and placenta accreta in women conceiving by in vitro fertilization. The preponderance of evidence implicated frozen embryo transfer cycles and, specifically, those employing programmed endometrial preparations, in the higher risk for these adverse maternal and neonatal pregnancy outcomes. Based upon this critical appraisal of the primary literature, we formulate potential etiologies and suggest strategies for prevention in the second article. METHODS: Comprehensive review of primary literature. RESULTS: Presupposing significant overlap of these apparently diverse pathological pregnancy outcomes within subjects who conceive by programmed autologous FET cycles, shared etiologies may be at play. One plausible but clearly provocative explanation is that aberrant decidualization arising from suboptimal endometrial preparation causes greater than normal trophoblast invasion and myometrial spiral artery remodeling. Thus, overly robust placentation produces larger placentas and fetuses that, in turn, lead to overcrowding of villi within the confines of the uterine cavity which encroach upon intervillous spaces precipitating placental ischemia, oxidative and syncytiotrophoblast stress, and, ultimately, late-onset or term preeclampsia. The absence of circulating corpus luteal factors like relaxin in most programmed cycles might further compromise decidualization and exacerbate the maternal endothelial response to deleterious circulating placental products like soluble fms-like tyrosine kinase-1 that mediate disease manifestations. An alternative, but not mutually exclusive, determinant might be a thinner endometrium frequently associated with programmed endometrial preparations, which could conspire with dysregulated decidualization to elicit greater than normal trophoblast invasion and myometrial spiral artery remodeling. In extreme cases, placenta accreta could conceivably arise. Though lower uterine artery resistance and pulsatility indices observed during early pregnancy in programmed embryo transfer cycles are consistent with this initiating event, quantitative analyses of trophoblast invasion and myometrial spiral artery remodeling required to validate the hypothesis have not yet been conducted. CONCLUSIONS: Endometrial preparation that is not optimal, absent circulating corpus luteal factors, or a combination thereof are attractive etiologies; however, the requisite investigations to prove them have yet to be undertaken. Presuming that in ongoing RCTs, some or all adverse pregnancy outcomes associated with programmed autologous FET are circumvented or mitigated by employing natural or stimulated cycles instead, then for women who can conceive using these regimens, they would be preferable. For the 15% or so of women who require programmed FET, additional research as suggested in this review is needed to elucidate the responsible mechanisms and develop preventative strategies.

Effects of long-acting injectable progesterone supplementation at early dioestrus on pregnancy maintenance in beef and dairy recipient cattle.

We tested in the present study the hypothesis that supplementation with long-acting P4 (iP4) at different times of the initial dioestrus improves pregnancy rates in dairy and beef recipients submitted to fixed-time embryo transfer (FTET). Recipients from commercial farms had their oestrous cycle synchronized with an E2/P4-based protocol in three experiments (Exp. 1 to 3). In Exp. 1, dairy heifers (n = 76) and cows (n = 104) were randomly assigned to two experimental groups: the control group (n = 89) and the iP4D4 group (n = 91). For Exps. 2 and 3, suckled beef recipients were used. In Exp. 2, recipients were assigned to two experimental groups: Control group (n = 147) and iP4D7 group (n = 144); whereas in Exp. 3, recipients were randomly assigned to three experimental groups: Control group (n = 85), iP4-D4 group (n = 86) and iP4D7 group (n = 81). Recipients in the iP4D4 and iP4-D7 groups received an i.m. administration of 150 mg iP4, on D4 or D7 (D0 was the day of expected oestrus). On D7, all recipients were evaluated by transrectal ultrasonography and those that had a CL received a fresh or vitrified in vitro-produced embryo. In Exp. 2 and 3, the CL area was also determined by ultrasonography at the time of FTET. The pregnancy diagnosis was performed at 30 days in Exp. 1, 57 days in Exp. 2, and between 40 and 72 days of pregnancy in Exp. 3. In Exp. 1, the pregnancy rate did not differ (p > .1) between the Control group (38.2% [34/89]) and iP4D4 group (49.5% [45/91]); yet, a parity effect indicated a greater (p < .05) pregnancy rate in heifers (57.9% [44/76]) than cows (30.8% [32/104]). In Exp. 2, the pregnancy rate was greater (p < .05) in the iP4D7 group (45.0% [65/144]) than in the Control group (34.0% [50/147]). Also, a greater (p = .08) pregnancy rate was observed for recipients with a small CL (≤2.75 cm2 ) that were treated with iP4 on the day of FTET than the control recipients (46.4% [32/69] vs. 32.6% [28/86]). In Exp. 3, no significant effects (p > .1) of the treatment group or CL size were detected on pregnancy rates at days 30 and 60. In conclusion, the beneficial effects of iP4 supplementation at early dioestrus on pregnancy maintenance may vary according to the experimental conditions, but its use at the time of FTET can be used as an alternative to enhance the fertility of beef recipients in challenging conditions in commercial herds.

Visfatin Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy.

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

TRPV2, a novel player in the human ovary and human granulosa cells.

The cation channel 'transient receptor potential vanilloid 2' (TRPV2) is activated by a broad spectrum of stimuli, including mechanical stretch, endogenous and exogenous chemical compounds, hormones, growth factors, reactive oxygen species, and cannabinoids. TRPV2 is known to be involved in inflammatory and immunological processes, which are also of relevance in the ovary. Yet, neither the presence nor possible roles of TRPV2 in the ovary have been investigated. Data mining indicated expression, for example, in granulosa cells (GCs) of the human ovary in situ, which was retained in cultured GCs derived from patients undergoing medical reproductive procedures. We performed immunohistochemistry of human and rhesus monkey ovarian sections and then cellular studies in cultured GCs, employing the preferential TRPV2 agonist cannabidiol (CBD). Immunohistochemistry showed TRPV2 staining in GCs of large antral follicles and corpus luteum but also in theca, endothelial, and stromal cells. TRPV2 transcript and protein levels increased upon administration of hCG or forskolin. Acutely, application of the agonist CBD elicited transient Ca2+ fluxes, which was followed by the production and secretion of several inflammatory factors, especially COX2, IL6, IL8, and PTX3, in a time- and dose-dependent manner. CBD interfered with progesterone synthesis and altered both the proteome and secretome, as revealed by a proteomic study. While studies are somewhat hampered by the lack of highly specific TRPV2 agonist or antagonists, the results pinpoint TRPV2 as a modulator of inflammation with possible roles in human ovarian (patho-)physiology. Finally, as TRPV2 is activated by cannabinoids, their possible ovarian actions should be further evaluated.

Progesterone signaling in the regulation of luteal steroidogenesis.

The corpus luteum is the major source of progesterone, the essential hormone for female reproductive function. While progesterone activity has been the subject of extensive research for decades, characterization of non-canonical progesterone receptor/signaling pathways provided a new perspective for understanding the complex signal transduction mechanisms exploited by the progesterone hormone. Deciphering these mechanisms has significant implications in the management of luteal phase disorders and early pregnancy complications. The purpose of this review is to highlight the complex mechanisms through which progesterone-induced signaling mediates luteal granulosa cell activity in the corpus luteum. Here, we review the literature and discuss the up-to-date evidence on how paracrine and autocrine effects of progesterone regulate luteal steroidogenic activity. We also review the limitations of the published data and highlight future research priorities.

Timing of the luteal-placental shift is delayed with additional fetuses in litter-bearing callitrichid monkeys, Saguinus oedipus and Callithrix jacchus.

The luteal-placental shift is an important milestone of mammalian pregnancy signifying when endocrine control of pregnancy shifts from the corpus luteum of the ovary to the placenta. The corpus luteum is maintained by chorionic gonadotropin (CG). Upon sufficient placental maturation, CG production wanes, the corpus luteum involutes, and control is shifted to the placenta, one consequence of which is a midgestational rise in glucocorticoid production, especially cortisol and cortisone, by both mother and fetus. Glucocorticoids are involved in initiating parturition, prenatal programming of offspring phenotype, and maturing fetal organs. Limited evidence from human pregnancy suggests that the timing of this shift is delayed in twin pregnancies, but little is known about the timing of the luteal-placental shift in litter-bearing monkeys from the primate family Callitrichidae. Here we provide evidence from cotton-top tamarins (Saguinus oedipus) and common marmosets (Callithrix jacchus) of longer duration of elevated CG associated with multiple infant births compared to single births. Urinary profiles from cotton-top tamarins demonstrate that the decline of the extended elevation of CG precedes the onset of the midpregnancy sustained rise in glucocorticoids; this shift occurs later with an increase from one to two fetuses carried to term. In the common marmoset, the onset of the sustained rise of glucocorticoids in maternal urine is also delayed with an increase in infant number. Total urinary glucocorticoid levels during the last half of gestation increase monthly but do not differ by infant number. The significant delay in the luteal-placental shift suggests a longer period of placental maturation is needed to support a greater number of fetuses.

Relationship between ovarian ultrasonographic findings on the seventh post-estrus day and plasma progesterone concentration, nutritional metabolic factors, and pregnancy outcome in dairy cows.

To improve the accuracy of ultrasonographic assessment of luteal function, we investigated the relationship between ovarian ultrasonographic findings on Day 7 (Day 1 = ovulation) and plasma progesterone (P4) concentration, nutritional metabolic factors, and pregnancy outcome. A total of 47 spontaneous estrus events were investigated in 38 lactating Holstein cows (artificial insemination, n = 31; embryo transfer, n = 16). Transrectal ultrasonography was performed on Days 0 and 7 to measure the pre-ovulatory follicle area on Day 0 and the luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (rLBF) (= LBF/LTA), and dominant follicle area (DFA) on Day 7. Blood samples were collected on Day 7 to measure plasma P4, insulin-like growth factor-I (IGF-I), insulin, and metabolites. Plasma P4 concentration was positively correlated with LTA but was not associated with LBF or rLBF. Plasma P4 concentration was positively correlated with blood glucose and IGF-I and negatively correlated with blood urea nitrogen and free fatty acid, and no significant relationship was found between the ultrasonographic findings of the corpus luteum (CL) and these blood metabolites. Pregnant cows had smaller DFA than non-pregnant cows. In conclusion, LTA measurement can help predict plasma P4 concentration, but it was difficult to detect variations in plasma P4 concentration in relation to changes in energy status by evaluating the CL ultrasonographically. A combined assessment of CL and first-wave dominant follicle may be important in evaluating fertility.