Hemoglobin: a Gas Transport Molecule That Is Hormonally Regulated in the Ovarian Follicle in Mice and Humans1

Abstract

An increasing number of nonerythroid tissues are found to express hemoglobin mRNA and protein. Hemoglobin is a well-described gas transport molecule, especially for O2, but also for NO, CO2, and CO, and also acts as a reactive oxygen species scavenger. We previously found Hba-a1 and Hbb mRNA and protein at high levels within mouse periovulatory cumulus cells, but not in cumulus following in vitro maturation. This led us to investigate the temporal and spatial regulation in follicular cells during the periovulatory period. Cumulus-oocyte complexes were collected from equine chorionic gonadotropin/human chorionic gonadotropin-treated peripubertal SV129 female mice and collected and analyzed for gene expression and protein localization at a variety of time points over the periovulatory period. A further cohort matured in vitro with different forms of hemoglobin (ferro- and ferrihemoglobin) under different O2 atmospheric conditions (2%, 5%, and 20% O2) were subsequently fertilized in vitro and cultured to the blastocyst stage. Murine mRNA transcripts for hemoglobin were regulated by stimulation of the ovulatory cascade, in both granulosa and cumulus cells, and expression of HBA1 and HBB was highly significant in human granulosa and cumulus, but erythrocyte cell marker genes were not. Several other genes involved in hemoglobin function were similarly luteinizing hormone-regulated, including genes for heme biosynthesis. Immunohistochemistry revealed a changing localization pattern of HBA-A1 protein in murine cumulus cells and oocytes following the ovulatory signal. Significantly, no positive staining for HBA-A1 protein was observed within in vitro-matured oocytes, but, if coincubated with ferro- or ferrihemoglobin, cytoplasmic HBA-A1 was observed, similar to in vivo-derived oocytes. Addition of ferro-, but not ferrihemoglobin, had a small, positive effect on blastocyst yield, but only under either 2% or 20% O2 gas atmosphere. The identification of hemoglobin within granulosa and cumulus cells poses many questions as to its function in these cells. There are several possible roles, the most likely of which is either an O2 or NO sequestering molecule; perhaps both roles are engaged. The strong endocrine regulation during the periovulatory period suggests to us that one potential function of hemoglobin is to provide a short-lived hypoxic environment by binding very tightly any available O2. This, in turn, facilitates the differentiation of the follicle towards corpus luteum formation by enabling the stabilization of a key transcription factor known to initiate such differentiation: hypoxia inducible factor.

The Ovarian Antral Follicle: Living on the Edge of Hypoxia or Not?1

“…To rule out suspected blood contamination during collection and tissue processing, we found very low to undetectable transcript abundance for erythrocyte gene markers (Gata1, Ahsp and Slc4a1). Furthermore, we determined that follicular transcripts for Hba-a1 and Hbb are hormonally regulated, being upregulated around the time of ovulation (12 h post hCG), then declining to pre-hCG administration levels [42]. We have also found transcripts for heme biosynthesis, which is unsurprising given the necessity of heme for other follicular enzymes, such as the cytochromes, but supports the possibility that active O 2 -binding hemoglobin may be assembled.…”

“…Following the LH surge, despite the increased vascular support and vascular permeability to the follicle, dissolved O 2 falls, hemoglobin (HBA1, HBB) within the follicle increases, and 2, 3-bisphosphoglycerate decreases (decreased activity of 2, 3-bisphosphoglycerate mutase, BPGM), causing a higher binding affinity for O 2 by hemoglobin, decreasing the granulosa/cumulus cell intracellular levels of follicular oxygen, thus triggering an increase in HIF-mediated VEGF induction. Taken from Brown et al [42] with permission. …”

“…It has been reported that the pO 2 of the follicle decreases during the peri-ovulatory period [54]. This coincides with an increase in follicular hemoglobin mRNA and protein levels, concurrently with a decrease in the abundance of the bisphosphoglycerate mutase mRNA levels, the enzyme that regulates 2,3-BPG levels [42]. The interaction between hemoglobin-bound O 2 and allosteric effector 2,3-BPG is a very well characterized functional modification.…”

“…We have also found transcripts for heme biosynthesis, which is unsurprising given the necessity of heme for other follicular enzymes, such as the cytochromes, but supports the possibility that active O 2 -binding hemoglobin may be assembled. Furthermore, peri-ovulatory human granulosa and cumulus cells had similarly high HBA1 and HBB transcript levels, but not of GATA1 [42]. Other non-hematopoietic tissues are also known to express hemoglobin, such as several neural cell types [43], hepatocytes [44], mesangial cells [45] macrophages [46], the eye's cornea [47] and retina [48].…”

An Intersectional Study of LncRNAs and mRNAs Reveals the Potential Therapeutic Targets of Buyang Huanwu Decoction in Experimental Intracerebral Hemorrhage

“…Hemoglobin (Hb) is a gas transporter with two α chains (hemoglobin alpha [HBA-A1]) and two β chains (hemoglobin beta [HBB-B1]). Each chain contains a heme subunit that binds to and delivers oxygen; four oxygen molecules are transported per molecule of hemoglobin [30, 31]. After ICH, high levels of Hb at lesions usually arise from the following 2 sources: the release of a large fraction of Hb from the hematoma after red blood cell lysis [32, 33] and the expression of the non-erythroid fraction of Hb in neurons and activated macrophages in response to hypoxia inducible factor-1 (HIF-1α) and erythropoietin (EPO) [34-36].…”