Physiological responses of Holstein calves and heifers carrying the SLICK1 allele to heat stress in California and Florida dairy farms.

Inheritance of the SLICK1 allele of the prolactin receptor gene improves thermotolerance of lactating Holstein cows under humid heat stress conditions. The aim of this study was to investigate whether pre- and postweaning Holstein heifers carrying the SLICK1 allele would show physiological responses indicative of higher tolerance to heat stress in high- and low-humidity climates. A total of 101 heifer calves of two age groups heterozygous for the SLICK1 allele and 103 wild-type half-siblings were evaluated during July 2020 in 3 dairy farms in central California and 2 in south Florida. Dry bulb temperature and relative humidity data were recorded during evaluation and used to calculate the temperature-humidity index (THI). Physiological measurements were obtained between 1600 and 1900 h in California, and 1200 and 1400 h in Florida and included rectal temperature, respiration rate, skin temperature, and sweating rate. Data were analyzed via Generalized Linear Mixed Models including the main effects of genotype, state, group, sire, farm within state, and interactions, with THI included as a covariate. The correlations between THI and dependent variables were analyzed via linear regression. The average 24-h THI was higher in Florida compared with California (90 vs. 72, respectively); the main driver of the higher THI in Florida was the high relative humidity (average 85.6% in Florida vs. 36.7% in California). In Florida, the rectal temperature of slick calves was 0.4°C lower than non-slick calves (39.5 ± 0.1 vs 39.9 ± 0.1°C); no differences were detected between slick and non-slick calves in California. Regardless of genotype, heifer calves in Florida had higher respiration rate, higher rectal and skin temperatures, and lower sweating rate than in California. This study is the first to evaluate physiological responses of calves carrying the SLICK1 allele under heat stress conditions in different climates. Our findings demonstrate that the presence of this allele is associated with lower rectal temperatures in pre- and post-weaning Holstein females. According to the physiological parameters evaluated, calves raised in Florida appeared to be under more severe heat stress; in those conditions, the SLICK1 allele was advantageous to confer thermotolerance as evidenced by lower rectal temperature in slick animals.

Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep.

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number.

Preantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

Gametes from stem cells: Status and applications in animal reproduction.

In vitro gamete differentiation could revolutionize animal production by decreasing generation intervals, increasing the number of gametes per animal and facilitating the dissemination of elite genetics. In addition, it could help to develop new strategies for the conservation of endangered species. The recent in vitro reconstitution of germ cell development in mice has inspired researchers to invest their best efforts into reproducing this achievement in livestock species. With this goal in mind, multiple differentiation approaches and cell sources have been evaluated. The degree of success in these evaluations varies according to the species and the stage of development studied, but, in general, partially positive results have been obtained. Evidence suggests that although functional gametes with true reproductive potential are still to be obtained, it is a matter of time before this goal is achieved.

Colony-stimulating factor 2 acts from days 5 to 7 of development to modify programming of the bovine conceptus at day 86 of gestation†.

Colony-stimulating factor 2 (CSF2) is an embryokine that improves competence of the embryo to establish pregnancy and which may participate in developmental programming. We tested whether culture of bovine embryos with CSF2 alters fetal development and alleviates abnormalities associated with in vitro production (IVP) of embryos. Pregnancies were established by artificial insemination (AI), transfer of an IVP embryo (IVP), or transfer of an IVP embryo treated with 10 ng/ml CSF2 from day 5 to 7 of development (CSF2). Pregnancies were produced using X-sorted semen. Female singleton conceptuses were collected on day 86 of gestation. There were few morphological differences between groups, although IVP and CSF2 fetuses were heavier than AI fetuses. Bicarbonate concentration in allantoic fluid was lower for IVP than for AI or CSF2. Expression of 92 genes in liver, placenta, and muscle was determined. The general pattern for liver and placenta was for IVP to alter expression and for CSF2 to sometimes reverse this effect. For muscle, CSF2 affected gene expression but did not generally reverse effects of IVP. Levels of methylation for each of the three tissues at 12 loci in the promoter of insulin-like growth factor 2 (IGF2) and five in the promoter of growth factor receptor bound protein 10 were unaffected by treatment except for CSF2 effects on two CpG for IGF2 in placenta and muscle. In conclusion, CSF2 can act as a developmental programming agent but alone is not able to abolish the adverse effects of IVP on fetal characteristics.

Association of single nucleotide polymorphisms in candidate genes previously related to genetic variation in fertility with phenotypic measurements of reproductive function in Holstein cows.

Many genetic markers related to health or production traits are not evaluated in populations independent of the discovery population or related to phenotype. Here we evaluated 68 single nucleotide polymorphisms (SNP) in candidate genes previously associated with genetic merit for fertility and production traits for association with phenotypic measurements of fertility in a population of Holstein cows that was selected based on predicted transmitting ability (PTA) for daughter pregnancy rate (DPR; high, ≥1, n = 989; low, ≤ -1.0, n = 1,285). Cows with a high PTA for DPR had higher pregnancy rate at first service, fewer services per conception, and fewer days open than cows with a low PTA for DPR. Of the 68 SNP, 11 were associated with pregnancy rate at first service, 16 with services per conception, and 19 with days open. Single nucleotide polymorphisms in 12 genes (BDH2, BSP3, CAST, CD2, CD14, FUT1, FYB, GCNT3, HSD17B7, IBSP, OCLN, and PCCB) had significant associations with 2 fertility traits, and SNP in 4 genes (CSPP1, FCER1G, PMM2, and TBC1D24) had significant associations with each of the 3 traits. Results from this experiment were compared with results from 2 earlier studies in which the SNP were associated with genetic estimates of fertility. One study involved the same animals as used here, and the other study was of an independent population of bulls. A total of 13 SNP associated with 1 or more phenotypic estimates of fertility were directionally associated with genetic estimates of fertility in the same cow population. Moreover, 14 SNP associated with reproductive phenotype were directionally associated with genetic estimates of fertility in the bull population. Nine SNP (located in BCAS, BSP3, CAST, FUT1, HSD17B7, OCLN, PCCB, PMM2, and TBC1D24) had a directional association with fertility in all 3 studies. Examination of the function of the genes with SNP associated with reproduction in more than one study indicates the importance of steroid hormones and immune function as determinants of reproductive function. All but 1 of the 68 evaluated SNP were variable in 11 breeds besides Holstein, indicating the potential effects of these SNP on reproductive function across breeds of cattle.

Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development.

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle.

The microenvironment of a preimplantation embryo can influence changes in development that affect postnatal phenotypes. One of the potential mediators of this effect in many species is colony-stimulating factor (CSF2), which can increase an embryo's ability to establish pregnancy after its transfer into recipients. Exposure of embryos to CSF2 during early development can also affect the pattern of development later in pregnancy in a sex-dependent manner. We therefore hypothesized that treatment of in vitro-produced embryos with CSF2 in culture would alter birth weight and postnatal growth of the resultant calf. Body weight and withers height were measured for Holstein heifer calves produced in vitro with or without 10 ng/ml CSF2 and for calves produced by artificial insemination. There were no differences in birth weight between groups; thereafter, however, calves from the CSF2-treated group experienced greater increases in body weight through 13 months of age, with only small differences in withers height. These results support the model that an embryo's postnatal characteristics can be programmed during the preimplantation period, and that CSF2 is one of the embryokines through which programming is directed. Mol. Reprod. Dev. 82: 892-897, 2015. © 2015 Wiley Periodicals, Inc.

The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo.

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.

Assessment of ovarian tissue and follicular integrity after cryopreservation via slow freezing or vitrification followed by in vitro culture.

OBJECTIVE: To evaluate ovarian tissue and follicle integrity before and after slow freezing or vitrification and postthawing in vitro culture. DESIGN: A laboratory study using bovine ovarian cortical tissue. SETTING: Academic laboratory. ANIMALS: Ovaries from healthy cattle. INTERVENTIONS: Bovine ovarian cortical tissue was subjected to either slow freezing or vitrification and subsequent in vitro culture. Tissue and follicle integrity were assessed before and after cryopreservation and culture. MAIN OUTCOME MEASURES: Hematoxylin and eosin staining was used to assess follicle stages, morphology, and stromal cell density. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to examine apoptosis, and Masson's trichrome staining was used to evaluate collagen content in the stromal environment. Immunofluorescent labeling was used to localize and quantify connexin 37 (CX37) and Ki67 expression. RESULTS: Regardless of previous cryopreservation, ovarian tissue culture resulted in a decreased percentage of primordial follicles and an increased percentage of primary follicles compared with fresh tissue, indicating that follicle activation was not negatively affected by cryopreservation. However, both culture and cryopreservation followed by culture decreased the percentage of normal preantral follicles compared with fresh tissue that had not been cultured. Culture and/or cryopreservation did not impact stromal cell number, but there was increased cell apoptosis in tissue that was cultured after vitrification compared with tissue that was not cultured. Tissue culture, regardless of cryopreservation, resulted in decreased collagen deposition. There were fewer follicles expressing CX37 in vitrified and thawed tissue compared with all other treatments. Cryopreservation and/or culture of ovarian tissue did not change the percentage of follicles that contained Ki67-positive granulosa cells or the percentage of Ki67-positive granulosa cells within those follicles. CONCLUSION: Based on these data, we conclude that tissue cryopreservation followed by culture does not affect follicle activation and growth, but it decreases the proportion of viable follicles within the tissue. Slow freezing was superior to vitrification as indicated by a higher proportion of follicles with normal morphology, lower stromal cell apoptosis, and maintenance of CX37 expression postthawing and after culture.

Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles.

Purpose: Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment. Methods: Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate. Results: Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm ( LHX1 ) and early coelomic epithelium/bipotential gonad markers ( OSR1 , GATA4 , WT1 ). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker FOXF1 . PAX3 expression was not induced, indicating absence of the paraxial mesoderm lineage. Conclusions: Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here. CAPSULE SUMMARY: We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.

Review: Roles of follicle-stimulating hormone in preantral folliculogenesis of domestic animals: what can we learn from model species and where do we go from here?

The pituitary gonadotropin FSH is a glycoprotein critical for the development of ovarian follicles. Upon binding to its G protein-coupled membrane receptor located on the granulosa cells of ovarian follicles, FSH elicits a cascade of downstream intracellular responses to promote follicle growth, maturation and steroidogenic activity, leading to the acquisition of meiotic and developmental competence of the enclosed oocyte. The essential role of FSH for proper antral follicle development and fertility is indisputable; over the decades, increasing evidence has also pointed toward survival and growth-promoting effects elicited by FSH in earlier-stage preantral follicles, deeming these follicles FSH-responsive as opposed to the FSH-dependent antral follicles. Transgenic mouse models lacking GnRH1, Fshß or Fshr clearly demonstrate this difference by showing that, morphologically, preantral follicles develop to the secondary stage without FSH signaling; however, exogenous expression or administration of FSH to hormone-deficient mice promotes preantral follicle development, with more pronounced effects seen in earlier stages (i.e., primary follicles). In hypophysectomized sheep, FSH administration also promotes the growth of primary-stage preantral follicles. However, in vivo studies in this area are more challenging to perform in domestic animals compared to rodents, and therefore most of the research to date has been done in vitro. Here, we present the existing evidence for a role of FSH in regulating the growth and survival of preantral follicles from data generated in rodents and domestic animals. We provide an overview of the process of folliculogenesis, FSH synthesis and cellular signaling, and the response to FSH by preantral follicles in vivo and in vitro, as well as interactions between FSH and other molecules to regulate preantral folliculogenesis. The widespread use of FSH in ovarian stimulation programs for assisted reproduction creates a real need for a better understanding of the effects of FSH beyond stimulation of antral follicle growth, and more research in this area could lead to the development of more effective fertility programs. In addition to its importance as an agricultural species, the cow provides a desirable model for humans regarding ovarian stimulation due to similar timing of folliculogenesis and follicle size, as well as similar ovarian architecture. The refinement of minimally invasive methods to allow the study of preantral folliculogenesis in live animals will be critical to understand the short- and long-term effects of FSH in ovarian folliculogenesis.

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 µm cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 µm and 40 µm cell strainers. The contents retained in the 40 µm strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Exposure to non-esterified fatty acids in vitro results in changes in the ovarian and follicular environment in cattle.

Negative energy balance (NEB) in the postpartum period of dairy cows is associated with reduced fertility to insemination later in lactation. We hypothesized that elevated non-esterified fatty acids (NEFA) levels that occur during NEB result in accumulation of fatty acids within the ovarian tissue and preantral follicles, causing changes in ovarian gene expression that would indicate a response to injury. We performed ovarian cortex culture and oocyte maturation in medium containing a combination of palmitic, oleic and stearic acid (NEFA). Ovarian cortex was subjected to RNA sequencing and lipid content analysis via Nile Red staining and gas chromatography; oocytes were analyzed for maturation rate and mitochondrial mass and localization following in vitro maturation (IVM). Accumulation of lipids associated with the plasma membrane was increased in granulosa cells of preantral follicles exposed to NEFA in vitro; RNA sequencing revealed changes in biological functions associated with metabolic disease, stimulation of an inflammatory response, and reduction in glucose uptake. Oocyte maturation under high NEFA compromised nuclear, but not cytoplasmic maturation. These data demonstrate that exposure to NEFA in vitro affects the ovary, preantral follicles and cumulus-oocyte complexes, and provides further insight into the potential links between metabolic imbalance and infertility.

Effects of the SLICK1 mutation in PRLR on regulation of core body temperature and global gene expression in liver in cattle.

The SLICK1 mutation in bovine PRLR (c.1382del; rs517047387) is a deletion mutation resulting in a protein with a truncated intracellular domain. Cattle carrying at least one allele have a phenotype characterized by a short hair coat (slick phenotype) and increased resistance to heat stress. Given the pleiotropic nature of prolactin, the mutation may affect other physiological characteristics. The liver is one organ that could potentially be affected because of the expression of PRLR. The mutation is a dominant allele, and heterozygous animals have a similar hair coat to that of animals homozygous for the mutation. Present objectives were to determine whether inheritance of the SLICK1 mutation affects liver gene expression and if animals homozygous for the SLICK1 allele differ from heterozygotes in liver gene expression and regulation of body temperature during heat stress. In one experiment, rectal and ruminal temperatures were less for Holstein heifers that were heterozygous for the SLICK1 allele compared with wildtype heifers. There were 71 differentially expressed genes in liver, with 13 upregulated and 58 downregulated in SLICK1 heterozygotes. Among the ontologies characteristic of differentially expressed genes were those related to immune function and fatty acid and amino acid metabolism. In a prospective cohort study conducted with adult Senepol cattle, body temperature and hepatic gene expression were compared between animals heterozygous or homozygous for the SLICK1 mutation. There were no differences in ruminal temperatures between genotypes, rectal temperature was higher in animals homozygous for the SLICK1 mutation, and there was only one gene in liver that was differentially expressed. It was concluded that inheritance of the SLICK1 allele can exert functional changes beyond those related to hair growth although changes in liver gene expression were not extensive. Results are also consistent with the SLICK1 allele being dominant because there were few differences in phenotype between animals inheriting one or two copies of the allele.

Ovarian preantral follicles are responsive to FSH as early as the primary stage of development.

Follicle-stimulating hormone (FSH) is required for ovarian antral folliculogenesis and steroidogenesis, and there is increasing evidence that it may play critical roles in preantral follicle development. We hypothesized that preantral follicles begin responding to FSH as early as the primary stage of development. Our objectives were to establish whether the FSH receptor (FSHR) was expressed in bovine preantral follicles and to determine the effects of FSH in these follicles and the surrounding ovarian tissue. Preantral follicles were isolated from bovine ovaries and subjected to immunolocalization of FSHR. Ovarian cortical strips were cultured with FSH or vehicle for 2 or 4 days and subjected to RNA sequencing, hematoxylin/eosin staining and immunostaining for p42/44 MAPK. Finally, cortical strips were cultured for 4 days with FSH before Western blot analysis of total and phosphorylated p42/44 MAPK and total aromatase. We found greater FSHR labeling intensity per cell in preantral follicles at the primary stage compared to other stages (P < 0.05). FSH upregulated genes involved in energy metabolism and MAPK signaling and downregulated genes related to phagosome and allograft rejection in the ovarian cortex. Preantral follicles cultured in situ with FSH had greater expression of total p42/44 MAPK (P < 0.05), but no difference was detected in whole tissue Western blot for phosphorylated p42/44 MAPK or aromatase. We conclude that the FSHR is expressed in preantral follicles as early as the primary stage of development, and that FSH upregulates cell metabolism and activates MAPK signaling pathways in preantral follicles.

Heat stress impairs in vitro development of preantral follicles of cattle.

Although detrimental effects of heat stress on antral follicle development have been well studied, long-term effects - affecting the preantral follicle pool - are still largely unknown. The goal of this study was to evaluate effects of heat stress on growth, viability, gene expression and ATP production of preantral follicles of cattle. Follicles at the primary, early secondary and secondary stages were isolated from cattle ovaries and individually cultured while imposing physiological (CON; 38.5 °C) or intermittent heat stress (HS; 38.5 °C for 16 h and 41 °C for 8 h daily) conditions for 7 days. Individual follicles were subjected to real-time qPCR for determination of relative abundance of BAX, HSPA1A and SOD1 mRNA transcripts and evaluated for ATP production. Treatment for 7 days with intermittent HS decreased viability (P =  0.01) and diameter (P =  0.03) of preantral follicles. Relative abundances of BAX and HSPA1A mRNA transcripts were greater in follicles of the CON and HS groups that became non-viable during culture (P <  0.05); relative abundance of SOD1 mRNA transcript, however, was only greater in non-viable follicles of the HS group (P <  0.05), but not non-viable follicles of the CON group (P =  0.3). The ATP production was not different between viable follicles of the CON and HS group (P = 0.86). In conclusion, all stages of growing preantral follicles of cattle were susceptible to negative effects of heat stress. Follicles at the secondary stage of development were most sensitive, followed by early secondary and primary follicles.

The impact of endocrine-disrupting chemical exposure in the mammalian hypothalamic-pituitary axis.

The hypothalamic-pituitary axis (HP axis) plays a critical and integrative role in the endocrine system control to maintain homeostasis. The HP axis is responsible for the hormonal events necessary to regulate the thyroid, adrenal glands, gonads, somatic growth, among other functions. Endocrine-disrupting chemicals (EDCs) are a worldwide public health concern. There is growing evidence that exposure to EDCs such as bisphenol A (BPA), some phthalates, polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and biphenyls (PBBs), dichlorodiphenyltrichloroethane (DDT), tributyltin (TBT), and atrazine (ATR), is associated with HP axis abnormalities. EDCs act on hormone receptors and their downstream signaling pathways and can interfere with hormone synthesis, metabolism, and actions. Because the HP axis function is particularly sensitive to endogenous hormonal changes, disruptions by EDCs can alter HP axis proper function, leading to important endocrine irregularities. Here, we review the evidence that EDCs could directly affect the mammalian HP axis function.

Influence of Sex on Basal and Dickkopf-1 Regulated Gene Expression in the Bovine Morula.

Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.