Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice†.

The reproductive homeobox X-linked (Rhox) genes encode transcription factors that are expressed selectively in reproductive tissues including the testis, epididymis, ovary, and placenta. While many Rhox genes are expressed in germ cells in the mouse testis, only Rhox8 is expressed exclusively in the Sertoli cells during embryonic and postnatal development, suggesting a possible role of Rhox8 in embryonic gonad development. Previously, Sertoli cell-specific knockdown of RHOX8 resulted in male subfertility due to germ cell defects. However, this knockdown model was limited in examining the functions of Rhox8 as RHOX8 knockdown occurred only postnatally, and there was still residual RHOX8 in the testis. In this study, we generated new Rhox8 knockout (KO) mice using the CRISPR/Cas9 system. Sex determination and fetal testis development were apparently normal in mutant mice. Fertility analysis showed a low fecundity in Rhox8 KO adult males, with disrupted spermatogenic cycles, increased germ cell apoptosis, and reduced sperm count and motility. Interestingly, Rhox8 KO testes showed an increase in testis size with dilated seminiferous tubules and rete testis, which might be affected by efferent duct (ED) Rhox8 ablation dysregulating the expression of metabolism and transport genes in the EDs. Taken together, the data presented in this study suggest that Rhox8 in the Sertoli cells is not essential for sex determination and embryonic testis differentiation but has an important role in complete spermatogenesis and optimal male fertility.

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

Normal Ovarian Function in Subfertile Mouse with Amhr2-Cre-Driven Ablation of Insr and Igf1r.

Insulin receptor signaling promotes cell differentiation, proliferation, and growth which are essential for oocyte maturation, embryo implantation, endometrial decidualization, and placentation. The dysregulation of insulin signaling in women with metabolic syndromes including diabetes exhibits poor pregnancy outcomes that are poorly understood. We utilized the Cre/LoxP system to target the tissue-specific conditional ablation of insulin receptor (Insr) and insulin-like growth factor-1 receptor (Igf1r) using an anti-Mullerian hormone receptor 2 (Amhr2) Cre-driver which is active in ovarian granulosa and uterine stromal cells. Our long-term goal is to examine insulin-dependent molecular mechanisms that underlie diabetic pregnancy complications, and our conditional knockout models allow for such investigation without confounding effects of ligand identity, source and cross-reactivity, or global metabolic status within dams. Puberty occurred with normal timing in all conditional knockout models. Estrous cycles progressed normally in Insrd/d females but were briefly stalled in diestrus in Igf1rd/d and double receptor (DKO) mice. The expression of vital ovulatory genes (Lhcgr, Pgr, Ptgs2) was not significantly different in 12 h post-hCG superovulated ovaries in knockout mice. Antral follicles exhibited an elevated apoptosis of granulosa cells in Igf1rd/d and DKO mice. However, the distribution of ovarian follicle subtypes and subsequent ovulations was normal in all insulin receptor mutants compared to littermate controls. While ovulation was normal, all knockout lines were subfertile suggesting that the loss of insulin receptor signaling in the uterine stroma elicits implantation and decidualization defects responsible for subfertility in Amhr2-Cre-derived insulin receptor mutants.

The NR2F2-HAND2 signaling axis regulates progesterone actions in the uterus at early pregnancy.

Endometrial function is dependent on a tight crosstalk between the epithelial and stromal cells of the endometrium. This communication is critical to ensure a fertile uterus and relies on progesterone and estrogen signaling to prepare a receptive uterus for embryo implantation in early pregnancy. One of the key mediators of this crosstalk is the orphan nuclear receptor NR2F2, which regulates uterine epithelial receptivity and stromal cell differentiation. In order to determine the molecular mechanism regulated by NR2F2, RNAseq analysis was conducted on the uterus of PgrCre;Nr2f2f/f mice at Day 3.5 of pregnancy. This transcriptomic analysis demonstrated Nr2f2 ablation in Pgr-expressing cells leads to a reduction of Hand2 expression, increased levels of Hand2 downstream effectors Fgf1 and Fgf18, and a transcriptome manifesting suppressed progesterone signaling with an altered immune baseline. ChIPseq analysis conducted on the Day 3.5 pregnant mouse uterus for NR2F2 demonstrated the majority of NR2F2 occupies genomic regions that have H3K27ac and H3K4me1 histone modifications, including the loci of major uterine transcription regulators Hand2, Egr1, and Zbtb16. Furthermore, functional analysis of an NR2F2 occupying site that is conserved between human and mouse was capable to enhance endogenous HAND2 mRNA expression with the CRISPR activator in human endometrial stroma cells. These data establish the NR2F2 dependent regulation of Hand2 in the stroma and identify a cis-acting element for this action. In summary, our findings reveal a role of the NR2F2-HAND2 regulatory axis that determines the uterine transcriptomic pattern in preparation for the endometrial receptivity.

Vapor Cannabis Exposure Generationally Affects Male Reproductive Functions in Mice.

This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3×/day over a 10-day period. Subsequently, F0 males were bred with drug-naïve CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, although apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis.