Perturbations in Lineage Specification of Granulosa and Theca Cells May Alter Corpus Luteum Formation and Function

Anovulation is a major cause of infertility, and it is the major leading reproductive disorder in mammalian females. Without ovulation, an oocyte is not released from the ovarian follicle to be fertilized and a corpus luteum is not formed. The corpus luteum formed from the luteinized somatic follicular cells following ovulation, vasculature cells, and immune cells is critical for progesterone production and maintenance of pregnancy. Follicular theca cells differentiate into small luteal cells (SLCs) that produce progesterone in response to luteinizing hormone (LH), and granulosa cells luteinize to become large luteal cells (LLCs) that have a high rate of basal production of progesterone. The formation and function of the corpus luteum rely on the appropriate proliferation and differentiation of both granulosa and theca cells. If any aspect of granulosa or theca cell luteinization is perturbed, then the resulting luteal cell populations (SLC, LLC, vascular, and immune cells) may be reduced and compromise progesterone production. Thus, many factors that affect the differentiation/lineage of the somatic cells and their gene expression profiles can alter the ability of a corpus luteum to produce the progesterone critical for pregnancy. Our laboratory has identified genes that are enriched in somatic follicular cells and luteal cells through gene expression microarray. This work was the first to compare the gene expression profiles of the four somatic cell types involved in the follicle-to-luteal transition and to support previous immunofluorescence data indicating theca cells differentiate into SLCs while granulosa cells become LLCs. Using these data and incorporating knowledge about the ways in which luteinization can go awry, we can extrapolate the impact that alterations in the theca and granulosa cell gene expression profiles and lineages could have on the formation and function of the corpus luteum. While interactions with other cell types such as vascular and immune cells are critical for appropriate corpus luteum function, we are restricting this review to focus on granulosa, theca, and luteal cells and how perturbations such as androgen excess and inflammation may affect their function and fertility

Letrozole: A Steroid-Free Estrous Synchronization Method

Most bovine estrous synchronization protocols utilize progesterone plus estrogen to control ovulation timing. A drug that inhibits estrogen production (Letrozole) may be an alternative, steroid-free synchronization method (not yet commercially available). However, low estrogen can negatively affect the health of follicles/oocytes and impact fertility. To determine its effects, Letrozole was administered intramuscularly while tracking follicle growth and circulating hormones. Letrozole response was variable. Two of three cows experienced delayed luteolysis/ovulation and extended progesterone production. This preliminary data indicates that Letrozole treatment allows normal follicle progression but drug response may vary and little is known about effects on oocyte quality

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide

Perturbations in Lineage Specification of Granulosa and Theca Cells May Alter Corpus Luteum Formation and Function

Anovulation is a major cause of infertility, and it is the major leading reproductive disorder in mammalian females. Without ovulation, an oocyte is not released from the ovarian follicle to be fertilized and a corpus luteum is not formed. The corpus luteum formed from the luteinized somatic follicular cells following ovulation, vasculature cells, and immune cells is critical for progesterone production and maintenance of pregnancy. Follicular theca cells differentiate into small luteal cells (SLCs) that produce progesterone in response to luteinizing hormone (LH), and granulosa cells luteinize to become large luteal cells (LLCs) that have a high rate of basal production of progesterone. The formation and function of the corpus luteum rely on the appropriate proliferation and differentiation of both granulosa and theca cells. If any aspect of granulosa or theca cell luteinization is perturbed, then the resulting luteal cell populations (SLC, LLC, vascular, and immune cells) may be reduced and compromise progesterone production. Thus, many factors that affect the differentiation/lineage of the somatic cells and their gene expression profiles can alter the ability of a corpus luteum to produce the progesterone critical for pregnancy. Our laboratory has identified genes that are enriched in somatic follicular cells and luteal cells through gene expression microarray. This work was the first to compare the gene expression profiles of the four somatic cell types involved in the follicle-to-luteal transition and to support previous immunofluorescence data indicating theca cells differentiate into SLCs while granulosa cells become LLCs. Using these data and incorporating knowledge about the ways in which luteinization can go awry, we can extrapolate the impact that alterations in the theca and granulosa cell gene expression profiles and lineages could have on the formation and function of the corpus luteum. While interactions with other cell types such as vascular and immune cells are critical for appropriate corpus luteum function, we are restricting this review to focus on granulosa, theca, and luteal cells and how perturbations such as androgen excess and inflammation may affect their function and fertility

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype

Letrozole: A Steroid-Free Estrous Synchronization Method

Most bovine estrous synchronization protocols utilize progesterone plus estrogen to control ovulation timing. A drug that inhibits estrogen production (Letrozole) may be an alternative, steroid-free synchronization method (not yet commercially available). However, low estrogen can negatively affect the health of follicles/oocytes and impact fertility. To determine its effects, Letrozole was administered intramuscularly while tracking follicle growth and circulating hormones. Letrozole response was variable. Two of three cows experienced delayed luteolysis/ovulation and extended progesterone production. This preliminary data indicates that Letrozole treatment allows normal follicle progression but drug response may vary and little is known about effects on oocyte quality

Granulosa Cell Exposure to Excess Androgens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess

Within the UNL physiology herd, a group of cows have been identified with excess androgen (androstenedione, A4) in their dominant follicle (30 fold higher than controls) and a 17% reduction in calving rate, suggesting subfertility. Th e objective was to identify altered granulosa cell gene expression that could be preventing these cells from converting excess androgen into estrogen. Microarray analysis suggests these granulosa cells experience inhibited proliferation resulting in a reduced total population of cells. Improved understanding of the causes of this phenotype may provide beef producers with tools to identify potentially subfertile cattle and improve reproductive efficiency

Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article “A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity” (McFee et. al., 2021 [1]

Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article “A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity” (McFee et. al., 2021 [1]