ALTERATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR A (VEGFA) ISOFORM EXPRESSION RESULTS IN ABNORMAL GONADAL FUNCTION

In the female, vascular endothelial growth factor A (VEGFA) isoforms regulate follicle development and affect the initial primordial follicle pool. In male rodent testes, they are involved in the development of vasculature and seminiferous cords. The objective of the current study was to evaluate effects of Sertoli and Granulosa cellspecific production of VEGFA isoforms on vascular development and gonadal morphogenesis. We used a DMRT1-cre crossed to a floxed VEGFA mouse to determine potential phenotypes in male Sertoli-cell (VEGFA-DSertG) and female Granulosa-cell (VEGFA-DGranG) specific knockouts. In females, we found smaller ovarian weight, fewer corpus luteums (CLs) with some abnormal CL morphology, and 55% reduction in estrogen in the VEGFA-DGranG compared to controls. The number of days mated until parturition was 12 days longer in VEGFA-DGranG X VEGFA-DSertG pairs versus control females mating with control males. In males, the weight of body, testes and epididymides in VEGFA-DSertG mice were significantly reduced than those from the controls. The morphology of epididymis and prostate appeared to be different with less tubules and branching, respectively compared to controls. Serum testosterone levels in VEGFA-DSertG tended to be higher than those in controls. Bcl-2 mRNA and Bcl-2/Bax in VEGFA-DSertG testis tended to be higher than that in controls. Sin3a mRNA within testis was higher in those three VEGFA-DSertG males with high serum testosterone concentration than controls. Therefore, we conclude that lack of VEGFA in Granulosa and Sertoli cells alters reproductive organ morphogenesis, reduces gonadal and male accessory gland organ weights, alters steroidogenesis and creates sub-fertility in mice

VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo

KDR-LacZ-expressing cells are involved in ovarian and testis-specific vascular development, suggesting a role for VEGFA in the regulation of this vasculature

Our objectives were to evaluate kinase insert domain protein receptor (KDR)-β-galactosidase (LacZ) expression as a marker for vascular development during gonadal morphogenesis and to determine whether any novel non-angiogenic KDR-LacZ expression was present in mouse testes or ovaries. Gonads were collected from mice expressing LacZ driven by the Kdr promoter (KDRLacZ) from embryonic day 11 (E11) through postnatal day 60 (P60). At E11.5, mesonephric cells expressing KDR-LacZ seemed to migrate into the developing testis and surrounded developing seminiferous cords. Cells expressing KDR-LacZ appeared in the ovary with no apparent migration from the adjacent mesonephros, suggesting a different origin of endothelial cells. Testis organ cultures from E11 mice were treated with 8 μM VEGFR-TKI, a vascular endothelial growth factor A signal transduction inhibitor; subsequently, the amount of KDR-LacZ staining was reduced by 66%-99% (

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in Granulosa Cells Using \u3ci\u3epDmrt-1-Cre\u3c/i\u3e or \u3ci\u3eAmhr2-Cre\u3c/i\u3e Reduces Fertility by Arresting Follicular Development and by Reducing Litter Size in Female Mice

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa-/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa-/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P \u3c 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa-/- females (P \u3c 0.007). The time between the first and second parturitions was also increased for heterozygous females (P \u3c 0.04) and tended to be increased for pDmrt1-Cre;Vegfa-/- females (P \u3c 0.07). pDmrt1-Cre;Vegfa-/- females had smaller ovaries (P \u3c 0.04), reduced plasma estradiol (P \u3c 0.007), fewer developing follicles (P \u3c 0.008) and tended to have fewer corpora lutea (P \u3c 0.08). Expression of Igf1r was reduced (P \u3c 0.05); expression of Foxo3a tended to be increased (P \u3c 0.06); and both Fshr (P \u3c 0.1) and Sirt6 tended to be reduced (P \u3c 0.06) in pDmrt1-Cre;Vegfa-/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa-/- mice that required more time from mating to first parturition (P \u3c 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter. Includes 2 supplementary files

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in the Testes of Male Mice Causes Subfertility, Reduces Sperm Numbers, and Alters Expression of Genes That Regulate Undifferentiated Spermatogonia

Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa−/− (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P \u3c .02) and tended to take longer for all subsequent parturition intervals (9 days; P \u3c .07). Heterozygous males sired fewer pups per litter (P \u3c .03) and after the first litter took 10 days longer (P \u3c .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P \u3c .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P \u3c .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P \u3c .02), Bcl2:Bax (P \u3c .02), Neurog3 (P \u3c .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P \u3c .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility

Oocyte mRNA and Follicle Androgen Levels Associated with Fertility

The environment that the oocyte develops in (follicle) and the mRNA that is produced (mRNA abundance) during development were examined. Androgen levels within the follicle were higher in heifers (≤ 2 years) that never established a pregnancy compared to cows that stayed in the herd at least 3 years and had at least one successful pregnancy. These high androgen levels were associated with increased abundance of several candidate mRNAs in the cumulus-oocyte complex (COC), which includes the oocyte and somatic cells immediately surrounding the oocyte, isolated from the dominant follicle. The data suggest that androgen levels represent a marker for oocyte quality which could be used to select for females to retain in the herd

Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity

Heifers and cows that were culled from the herd due to failure to become pregnant were categorized into groups with low (\u3c 2 year), moderate (\u3e2 and \u3c 6 year) or high (≥ 6 year) fertility. Antral follicle counts were numerically lower in the low group and increased in the moderate- and high-fertility group. Granulosa cells from dominant follicles in moderate- and high-fertility cows had a greater ratio of Vascular Endothelial Growth Factor 164 (VEGF164) to VEGF164B compared to the low-fertility cows. Furthermore, there was more CARTPT in granulosa cells from subordinate follicles in moderate- and high-fertility cows than low. Gene expression is altered in granulosa cells from cows differing in fertility, suggesting these are candidate genes that may be used as markers to assist in determining reproductive longevity in beef cows